Photochemical evidence for the participation of histidine in the active center of carboxy-peptidase-Ay
by Kenneth Allen Freude
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY in Chemistry
Montana State University
© Copyright by Kenneth Allen Freude (1969)
Abstract:
Dye-sensitized photooxidation using either methylene blue or Rose Bengal as sensitizers rapidly
diminishes both the peptidase and esterase activities of carboxypeptidase-Aγ. It is significant that both
activities are simultaneously diminished by this chemical modification procedure, since heretofore a
dual inactivation has been accomplished only by removal of the metal from the enzyme. All other
chemical modifications employed on carboxypeptidase-A previously reported, including methylene
blue sensitized photooxidation of carboxypeptidase-Aδ have resulted in an increase in esterase activity
and a decrease in peptidase activity. There is a considerable difference in the mechanism of action of
Rose Bengal and methylene blue. Rose Bengal functions as a very effective sensitizer for
photooxidation of both carboxypeptidase-Aγ and apocarboxypeptidase-Aγ, while methylene blue
sensitizes the photooxidation of only the metal free form of the enzyme. Both sensitizers show a
marked pH dependence and give identical sigmoidal curves which are typical of the pH dependency of
the dye-sensitized photooxidation of the imidazole moiety. Crystal violet, a sensitizer which is reported
to be specific for cysteine residues, was found to be completely ineffective on
apocarboxypeptidase-Aγ. Amino acid analysis of photooxidized enzyme revealed the loss of only
histidine residues. Photosensitive amino acids such as cysteine, tyrosine, tryptophan, and methionine
remained essentially unaltered. Significant protection against photooxidation was demonstrated using
the substrates hippuryl-L-phenylalanine , glycyltyrosine, and the competitive inhibitor
g-phenylpropionate. Kinetic studies indicate that Rose Bengal also functions as a competitive inhibitor
of carboxypeptidase-Aγ and prevents the exchange of metal ions at the active center of the enzyme.
The photolability of several metallo derivatives of carboxypeptidase-Aγ was investigated.
Conformational changes subsequent to photooxidation and the photolability of the enzyme in various
buffer systems were also investigated. K e n n e th A l l e n
F re u d e
ALL RIGHTS RESERVED
1970
PHOTOCHEMICAL EVIDENCE FOR THE PARTICIPATION OF HISTIDINE
IN THE ACTIVE CENTER OF CARBOXYPEPTIDASE-Ay
by
KENNETH ALLEN FREUDE
tV
A t h e s i s s u b m itte d to th e G ra d u a te F a c u lty in p a r t i a l
fu lf illm e n t o f th e r e q u ire m e n ts for th e d e g re e
of
DOCTOR OF PHILOSOPHY
in '
C h e m is tr y
Approved:
H e a d , M a jo r D e p a rtm e n t
, / V nJinLg - Com m it
C h a i r m a n , Exa
. MONTANA STATE UNIVERSITY
B o zem an , .M o n tan a ■
M a r c h , 1969.
ACKNOWLEDGEMENT
I w i s h to e x p r e s s my th a n k s to Dr.- Sam uel J, R o g e r s , D r. G ordon
R. J u l i a n , a n d D r. John E . R o b b in s, for t h e i r i n t e r e s t , a n d s tim u la tin g
d i s c u s s i o n s d u rin g th e c o u r s e o f th is w o r k , a n d in th e p r e p a r a t io n o f the
m a n u s c r ip t .
I am p a r t i c u l a r l y g r a te f u l to my w if e , S h a r o n , for h e r
p a t i e n c e a n d u n d e r s ta n d in g d u rin g my g r a d u a te c a r e e r , a n d for h e r
i n d i s p e n s a b l e a id in p re p a rin g a n d ty p in g th e m a n u s c r ip t .
I w ish a lso
to e x p r e s s my th a n k s t o . t h e o th e r m em b ers o f th e C h e m is tr y s t a f f , whom
I h a v e com e to k n o w , for t h e i r e n c o u r a g e m e n t a n d c o u n s e l d u rin g my
g r a d u a te c a r e e r .
TABLE OF CONTENTS
PAGE
L IS T OF TABLES
■
L IS T OF FIGURES
ABSTRACT
v
v ii
ix
INTRODUCTION
I
MATERIALS AND METHODS
3
PROCEDURE AND RESULTS
6
DISCUSSION AND CONCLUSIONS
71
SUMMARY
91
LITERATURE CITED
92
LIST OF TABLES
TABLE
PAGE
I . -P h o to o x id a tio n o f C a r b d x y p e p ti d a s e -Ay in th e p r e s e n c e
o f I , IO -P h e n a n th ro lin e a n d m e th y le n e b lu e ....................... 7
II.
The e f f e c t o f P h o to o x id a tio n w ith m e th y le n e b lu e in th e
p r e s e n c e o f I , IO -P h e n a n th ro lin e on E s t e r a s e A c tiv ity . 8
I I I . . The e f f e c t o f m e th y le n e b lu e OP P h o to o x id a tio n o f
. C a r b o x y p e p ti d a s e -Ay on E s t e r a s e A c t i v i t y ............. ... . .10
IV.
P h o to o x id a tio n of C a r b o x y p e p ti d a s e - A y w ith i n c r e a s e d
m e th y le n e b lu e a n d Rose B engal C o n c e n tr a tio n s . . .
11
V. .In h ib itio n ., R e a c t i v a t i o n , a n d P h o to o x id a tio n of
C a r b o x y p e p t i d a s e - A y in th e p r e s e n c e o f . EDTA . . . . 'TB
VI. P h o t o s e n s i t i z a t i o n o f I , IO -P h e n a n th ro lin e . . . . . . . . . . .
.14
VII . P r e p a r a tio n o f A p oenzym e o n S e p h a d e x G -2 5 C olum n . . . .
.16
V III . R e a c tiv a tio n o f A p o c a r b o x y p e p tid a s e - A y w ith Zinc Io n s . . .
17
IX,
X.
P h o to o x id a tio n of A p o c a r b o x y p e p tid a s e - A y w ith
m e th y le n e b lu e
.................................... ...
19
P h o to o x id a tio n o f C a r b o x y p e p t i d a s e - A y a n d P e p t i d a s e
A c tiv ity
............. ......................... ...
.2 0
XI. Rate o f l o s s o f P e p t i d a s e A c tiv ity o f C a r b o x y p e p tid a s e - A y
w h e n P h o to o x id iz e d in p r e s e n c e o f Rose B engal . . . 21
XII.
Rate o f l o s s o f . E s t e r a s e A c tiv ity o f C a r b o x y p e p tid a s e - A y
w h e n P h o to o x id iz e d in p r e s e n c e o f Rose B engal . . . 23
XIII. Rose B engal P h o to o x i d a tio n , E s t e r a s e A c tiv ity . . . . . . . . .
XIV.
Rose B engal P h o to o x i d a tio n , .E s te ra s e A c tiv ity . . . . . . . .
.2 4
. 25
XV. Rose B engal P h o to o x i d a tio n , E s t e r a s e A c tiv ity .................... . .26
XVI.
Rose B engal P h o to o x i d y tio n , P e p t i d a s e A c tiv ity . . . . . . . . 27
XVII.
P h o to o x id a tio n o f C a r b o x y p e p ti d a s e - A y w ith C r y s ta l
V io le t S e n s i t i z e r ...........................................
28
—v i —
XVIII,
pH D e p e n d e n c e o f P h o to o x id a tio n o f C a r b o x y p e p tid a s e - A y
in th e p r e s e n c e o f I , IO -P h e n a n th ro lin e a n d
m e th y le n e b lu e .................................................... ......................... 30
XIX.
pH D e p e n d e n c e o f P h o to o x id a tio n o f C a r b o x y p e p tid a s e - A y
w ith Rose B engal a s S e n s i t i z e r ................ ...................... . . 3 2
XX.
E ffe c t o f .P e p tid a s e S u b s tr a te on r a te o f P h o to o x id a tio n
w ith R ose B engal . . . . . . . . . . . . . . . . . . . . . . . .
33
S u b s tr a te P r o t e c t i o n , Rose B engal S y ste m . . . . . . . . . . . .
34
XXI.
XXII.
P r o te c tio n a g a i n s t th e P h o to o x id a tio n b y H ip p u r y l- L P h e n y la la n in e . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 6
XXIII.
S u b s tr a te P r o te c tio n Rose B engal S y ste m . . . . . . . . . . . . .
37
XXIV.
P h o to o x id a tio n in th e p r e s e n c e o f p-P h en y lp ro p i o n a te . . . . . 39
XXV. P h o to o x id a tio n in th e p r e s e n c e o f |3-P h en y lp ro p io n ate . . . . . 40
XXVI.
G ly c y ity r o s in e P r o t e c t i o n .................................................... ...
XXVII.
Co
XXVIII.
2+
41
A c tiv a tio n o f P h o to o x id iz e d C a r b o x y p e p ti d a s e - A y . . . . 54
P h o to o x id a tio n o f C o b a l t - C a r b o x y p e p t i d a s e - A y a n d n a tiv e
C a r b o x y p e p t i d a s e - A y i n s a m e Buffer S y s t e m ....................5 6
XXIX
P r e p a r a tio n a n d P h o to o x id a tio n o f N i c k e l - C a r b o x y p e p t i d a s e . 57
XXX.
.C o p p e r C a r b o x y p e p ti d a s e - A y
59
E ffe c t o f R ose B engal on th e e x c h a n g e o f C u ^+ a n d Zn^+
io n in C a r b o x y p e p ti d a s e - A y . . . . . . . . . . . . . . . . .
61
P h o to o x id a tio n o f C a r b o x y p e p tid a s e - A y in th e p r e s e n c e
o f 10~3 M Cu2+ io n s . . . . . . . . . . . . . . . . . . . . . .
62
.XXXIII. .P h o to o x id a tio n o f C o b a l t - C a r b o x y p e p t i d a s e - A y . . . . . . . . .
66
XXXIV. P h o to o x id a tio n o f C a r b o x y p e p ti d a s e - A y in 0 ..05 M so d iu m
a c e t a t e , 0 .0 5 M T r i s , 1 .0 M N a C l Buffer . . . . . . . .
67
XXXI.
XXXII.
XXXV. P h o to o x id a tio n o f C a r b o x y p e p ti d a s e in A c e ta te Ion Buffer . . 68
XXXVI. P h o to o x id a tio n o f C a r b o x y p e p ti d a s e - A y in v a r io u s
Buffer S y s te m s .............................................. ...
70
LIST OF FIGURES
FIGURE
PAGE
I.
S e p h a d e x G -7 5 e l u tio n p a t t e r n s o f C a r b o x y p e p ti d a s e - A y
p h o to o x id iz e d for tw e n ty m i n u t e s ................................................. •„ 43
2.
S e p h a d e x G -7 5 e l u tio n p a t t e r n s o f C a r b o x y p e p ti d a s e - A y
p h o to o x id iz e d for s i x t y m i n u t e s ................................. ...
44
S e p h a d e x G -7 5 e l u tio n p a t t e r n s o f C a r b o x y p e p ti d a s e - A y
u n o x id iz e d . . . ........................................................... ...
45
3.
4 . .E lution p a t t e r n s o f u n o x id iz e d C a r b o x y p e p ti d a s e - A y on
TEAE c e l l u l o s e .............................
5.
6.
E lu tio n p a t t e r n s o f o x id iz e d C a r b o x y p e p ti d a s e on TEAE
c e l l u l o s e , p h o to o x id iz e d
for
tw e n ty m i n u t e s .
E lu tio n p a t t e r n s o f n a tiv e C a r b o x y p e p ti d a s e - A y on
S e p h a d e x G -7 5 c o l u m n .................
9 . . E lu tio n p a t t e r n s o f C a r b o x y p e p ti d a s e - A y on S e p h a d e x G -7 5
c o l u m n .................................................................................
10.
47
E lu tio n p a t t e r n s o f o x id iz e d C a r b o x y p e p ti d a s e on TEAE
c e l l u l o s e , p h o to o x id iz e d
for
th ir ty m i n u t e s .................... 48
7 . . E lu tio n p a t t e r n s o f u n o x id iz e d C a r b o x y p e p ti d a s e - A y on
TEAE c e l l u l o s e ..........................................................................................
8.
46
E lu tio n p a t t e r n o f C a r b o x y p e p ti d a s e - A y on S e p h a d e x G -7 5
co lu m n ....................................................
.11. .S e p h a d e x G -7 5 e l u tio n p a t t e r n s o f C a r b o x y p e p ti d a s e - A y in
th e p r e s e n c e o f 1 0 ~^ m C u ^+ i o n s ....................... ...
. 49
50
51
52
63
12.
S e p h a d e x G -7 5 e l u tio n p a t t e r n s o f C a r b o x y p e p ti d a s e - A y in
th e p r e s e n c e o f1 0 ~ 3 m C u ^+ io n s . . . . . . . . . . . . . . . . . . 64
13.
P h o to o x id a tio n w ith m e th y le n e b lu e in p r e s e n c e a n d a b s e n c e
o f 1 , 1 0 - p h e n a n t h r o l i n e ........................
72
L o s s o f p e p t i d a s e a n d e s t e r a s e a c t i v i t y a s a r e s u l t of
p h o t o o x i d a . t i o n ......................
73
14.
15. .M e th y le n e b lu e a n d R ose B engal a s e f f e c t iv e s e n s i t i z e r s in
th e p r e s e n c e a n d a b s e n c e o f 1 ,1 0 - p h e n a n th r o lin e ......................... 76
—
viii —
16.
17.
18.
2+
2+
The e f f e c t o f R ose B engal on th e e x c h a n g e o f Cu
a n d Zn
io n s in C a r b o x y p e p ti d a s e - A y . . . . . . . . . . . . . . . . . . . .
78
L in e w e a v e r-B u rk p lo t o f C a r b o x y p e p tid a s e a c t i v i t y in th e
p r e s e n c e a n d a b s e n c e o f Rose B engal . . . . . . . . . . . . . . .
79
S e m i- lo g a r ith m ic p lo t o f l o s s o f e s t e r a s e a c t i v i t y a s a
f u n c tio n o f tim e .................................................................
81
19. . The pH d e p e n d e n c e o f m e th y le n e b lu e -1 ,.1 0 -p h e n a n th ro lin e
a n d Rose B engal p h o to o x id a tio n o f C a r b o x y p e p ti d a s e - A y . . .83
2 0 . P r o te c tio n a g a i n s t Rose B engal p h o to o x id a tio n of
C a r b o x y p e p ti d a s e -Ay w ith th e s u b s t r a t e
h ip p u r y l - L - p h e n y l a l a n i n e . ' .............................................
21.
84
P r o te c tio n a g a i n s t Rose B engal p h o to o x id a tio n w ith
p -p h e n y lp ro p io n a te . ■................
84
2 2 . P r o te c tio n a g a i n s t Rose B engal p h o to o x id a tio n w ith
g l y c y l t y r o s i n e ..................................................
85
2 3. Amino a c i d a n a l y s i s o f o x id iz e d a n d u n o x id iz e d
C a r b o x y p e p ti d a s e - A y ................................................................
.89
ABSTRACT
D y e - s e n s i t i z e d p h o to o x id a tio n u s in g e i t h e r m e th y le n e b lu e or
R ose B engal a s s e n s i t i z e r s r a p id ly d i m in is h e s b o th th e p e p t i d a s e a n d
e s t e r a s e a c t i v i t i e s o f c a r b o x y p e p tid a s e - A y . It i s s i g n i f i c a n t t h a t b o th
a c t i v i t i e s a re s i m u l t a n e o u s l y d im in is h e d b y t h i s c h e m ic a l m o d if ic a tio n
p r o c e d u r e , s i n c e h e re to fo re a d u a l i n a c t i v a t i o n h a s b e e n a c c o m p lis h e d
o n ly b y re m o v a l o f th e m e ta l from th e e n z y m e . All o th e r c h e m ic a l
m o d if ic a tio n s e m p lo y e d on c a r b o x y p e p tid a s e - A p r e v io u s ly r e p o r t e d ,
in c lu d in g m e th y le n e b lu e s e n s i t i z e d p h o to o x id a tio n o f c a r b o x y p e p t i d a s e A§ hay d r e s u l t e d in an. i n c r e a s e in e s t e r a s e a c t i v i t y a n d a d e c r e a s e in
. p e p t i d a s e a c t i v i t y . There i s a c o n s i d e r a b l e d if f e r e n c e in. th e m e c h a n is m
o f a c t i o n o f Rose B engal a n d m e th y le n e b l u e . Rose B engal f u n c tio n s a s
a v e ry e f f e c t i v e s e n s i t i z e r for p h o to o x id a tio n o f b o th c a r b o x y p e p tid a s e - A
a n d a p o c a r b o x y p e p tid a s e - A y , w h ile m e th y le n e b lu e s e n s i t i z e s th e
p h o to o x id a tio n o f o n ly th e m e ta l fre e form o f th e e n z y m e . . Both s e n s i ­
t i z e r s s h o w a m a rk e d pH d e p e n d e n c e a n d g iv e i d e n t i c a l s ig m o id a l
c u rv e s w h ic h a re t y p i c a l o f th e pH d e p e n d e n c y o f th e d y e - s e n s i t i z e d
. p h o to o x id a tio n o f th e im id a z o le m o ie ty . C r y s ta l v i o l e t , a s e n s i t i z e r
w h ic h i s r e p o rte d to be s p e c i f i c for c y s t e i n e r e s i d u e s , w a s found to be
c o m p le te ly in e f f e c t i v e on a p o c a r b o x y p e p tid a s e - A y . Amino acid , a n a l y s i s
o f p h o to o x id iz e d e n z y m e r e v e a l e d th e l o s s o f o n ly h i s t i d i n e r e s i d u e s .
P h o t o s e n s i t i v e a m in o -a c id s s u c h a s c y s t e i n e , t y r o s i n e , t r y p t o p h a n , and
m e th io n in e re m a in e d e s s e n t i a l l y u n a l t e r e d . ■ S ig n if ic a n t p r o t e c t i o n
a g a i n s t p h o to o x id a tio n w a s d e m o n s t r a t e d u s in g th e s u b s t r a t e s h ip p u r y lL - p h e n y l a l a n i n e , g ly c y l ty ro s i n e , a n d thje ^com petitive in h ib ito r
g - p h e n y lp r o p io n a te . K in e tic s t u d i e s i n d i c a t e t h a t Rose B engal a l s o
fu n c tio n s a s a c o m p e titiv e in h ib ito r o f c a r b o x y p e p tid a s e - A y a n d p r e v e n ts
th e e x c h a n g e o f m e ta l io n s a t th e a c t i v e C enter o f th e e n z y m e . The
p h o t o l a b i l i t y o f s e v e r a l m e ta llo d e r i v a t i v e s o f c a r b o x y p e p tid a s e - A y w a s
i n v e s t i g a t e d . C o n fo rm a tio n a l c h a n g e s s u b s e q u e n t to p h o to o x id a tio n a n d
th e p h o to l a b i l i t y o f th e e n zy m e in v a r io u s b u ffe r s y s te m s w e re a l s o
in v e stig a te d .
INTRODUCTION
C a r b o x y p e p t i d a s e - A y i s a z i n c m e ta llo e n z y m e w h ic h e x h ib its
p e p tid a se and e s te ra s e a c t i v i t i e s . ^
The z i n c atom c a n b e r e v e r s i b l y
r e m o v e d , w h ic h r e s u l t s i n .t h e l o s s o f b o th th e p e p t i d a s e a n d e s t e r a s e
( 2)
a c tiv itie s.
S i n c e . t h e re m o v a l o f th e m e ta l io n from t h i s m e ta llo e n z y m e
i s r e v e r s i b l e , i t s h o u ld be p o s s i b l e to i d e n tif y th e m e ta l b in d in g s i t e by
e m p lo y in g v a r io u s s p e c i f i c c h e m ic a l r e a g e n t s or o th e r p r o c e d u r e s .
Such
c h e m ic a l m o d if ic a tio n p r o c e d u r e s h a v e le d to th e s u g g e s t i o n t h a t .the
m e ta l io n is b o u n d to a th io l group o f a c y s t e i n e r e s i d u e a n d to th e
Q/-amino group o f th e N - te r m in a l am ino a c i d o f c a rb o x y p e p tid a s e - A § .
(3)
I n te r p r e ta tio n o f th e r e s u l t s o f th e m e th o d s u s e d to d e t e c t th e
th io l group in t h i s e n z y m e a re s u b j e c t to q u e s t i o n .
R e a g e n ts u s e f u l
for th io l d e t e c t i o n , s u c h a s io d o and. b r o m o a c e t a t e , i o d o a c e ta m id e and
(3)
N - e th y lm a le im id e fa il to r e a c t w ith th e n a tiv e or m e ta l fre e e n z y m e .
S u lfh y d ry l g ro u p s w e r e , h o w e v e r , d e te r m in e d u s in g s i l v e r i o n , p - m e c u r i (3)
b e n z o a te , and fe rric y a n id e .
It i s i n t e r e s t i n g t h a t th e o n ly m e th o d s
w h ic h w e re c a p a b le o f d e m o n s tr a tin g th e p r e s e n c e of fre e s u lfh y d ry l
g ro u p s in th is e n zy m e s y s te m w e re m e th o d s w h ic h d e p e n d e d u p o n m e ta l
b in d in g .
T his f a c t b e c o m e s e x c e e d i n g l y im p o rta n t w h e n o n e c o n s id e r s
t h a t t h e s e m e th o d s w e re a p p lie d to a s y s te m w h ic h i s know n to bind
m e t a l s , t h a t is c a r b o x y p e p tid a s e - A y .
In a d d i t i o n , w h e n c a r b o x y p e p ti-
d a s e w a s s u b j e c t to r e d u c in g r e a g e n t s s u c h a s so d iu m b o r o h y d r id e , or
2 - m e r c a p t o e t h a n o l , i t w o u ld th e n form d e r i v a t i v e s w ith i o d o a c e t a t e and
N -e th y lm a le im id e .
T h e s e r e s u l t s im p ly t h a t th e e n z y m e c o n ta i n s in
i t s s tr u c tu r e a d is u lf id e l i n k a g e i n s t e a d of tw o c h e m i c a lly u n r e a c ti v e
(4)
s u lfh y d ry l g r o u p s .
C o m p le x o m e tric t i t r a t i o n ^ o f a p o c a r b o x y p e p t i d a s e w ith ' Zn^+
r e v e a l s tw o b in d in g g ro u p s o f a p p a r e n t pK ^1s o f 7 . 7 and. 9 . L
The pK&
o f 9 .1 r e p r e s e n t s th e o n ly s u b s t a n t i a l e v id e n c e o f.th e in v o lv e m e n t of
s u lf h y d r y l, b u t b y no m e a n s co n firm s t h i s h y p o t h e s i s .
The pKQ of .7.7
w o u ld c o r r e s p o n d to e i t h e r . t h e t i t r a t a b l e p ro to n o f im id a z o le o r o f th e
N - te r m in a l Q,-amino .g ro u p .
The in v o lv e m e n t o f im id a z o le s e e m s a
b e t t e r c h o ic e for th e r e a s o n s s u g g e s t e d h e r e .
C a r b o x y p e p ti d a s e
-2 -
e x i s t s in th r e e m a jo r forms a n d one m in o r fo r m .
T h e se forms , th e a l p h a ,
b e t a , g a m m a , a n d d e l t a , h a v e i d e n t i c a l s p e c i f i c i t i e s , b u t d iff e r in
s o l u b i l i t y , i n . c h a i n l e n g t h , a n d in t h e i r N - te r m in a l r e s i d u e s .
The
N - te r m in a l r e s i d u e s a re a l a n i n e , s e r i n e , a s p a r a g i n e , a n d a s p a r a g i n e ,
re sp ec tiv e ly .
D ue to th e h e t e r o g e n i e t y o f .th e N - te r m in a l re g io n o f th e
m o l e c u l e , c o n fo rm a tio n a l d i f f e r e n c e s a m o n g th e v a r io u s forms w o u ld b e
e x p e c t e d i f th e binding, o f z in c a to m s in v o lv e d p a r t i c i p a t i o n o f th e
N - te r m in a l r e s i d u e s .
T h e s e c o n fo rm a tio n a l d i f f e r e n c e s s h o u ld r e s u l t
in a l t e r e d s p e c i f i c i t y o f th e v a r io u s e n z y m a tic f o r m s , which, i s no t
observed.
/
m e ta l. '
F u r t h e r , p r o c a r b o x y p e p t id a s e h a s b e e n sh o w n to b in d
n\
■ P r o c a r b o x y p e p tid a s e c o n s i s t s o f a s i n g le p o ly p e p tid e c h a in
o f m o le c u la r w e ig h t 8 7 , 0 0 0 , a n d y ie ld s o n e o f th e four c a r b o x y p e p tid a s e s - A , u p o n c l e a v a g e b y th e e n d o p e p t i d a s e s t r y p s i n a n d c h y m o try p s in .
This f a c t i n d i c a t e s t h a t th e b in d in g s i t e of th e z in c is a l r e a d y e s t a b ­
l i s h e d in th e p r o c a r b o x y p e p t i d a s e , a n d t h a t th e N - te r m in u s o f
c a r b o x y p e p t i d a s e is n o t in v o lv ed , in z in c b i n d i n g .
The p o s s i b l e
in v o lv e m e n t o f im id a z o le in th e c a t a l y t i c a c t i v i t y of c a r b o x y p e p tid a s e
h a s been, im p lie d in s e v e r a l s t u d i e s .
S in c e th e r e w a s c o n s i d e r a b l e e v id e n c e in d i c a t i n g t h a t h i s t i d i n e
c o u ld be in v o lv e d in th e a c t i v i t y o f c a r b o x y p e p t i d a s e , a p h o to c h e m ic a l
i n v e s t i g a t i o n w a s u n d e r ta k e n on th e gam m a form o f th e e n z y m e .
MATERIALS AND METHODS
The p e p t i d a s e a n d e s t e r a s e a c t i v i t i e s w e re d e te r m in e d in th e
fo llo w in g m anner;
The rate- o f h y d r o ly s is o f h i p p u r y l - L - p h e n y l a l a n i n e ,
. (HPA), th e p e p tid e s u b s t r a t e , or o f h i p p u r y l - D L - p h e n y l l a c t i c a c i d ,
(HPLA), th e e s t e r a s e s u b s t r a t e , w a s m e a s u r e d b y th e i n c r e a s e in
a b s o r b a n c e a t 25,4 mp, a t 2 5 ° C . A u n it o f a c t i v i t y i s e q u a l to one
m icro m o le o f s u b s t r a t e h y d ro ly z e d p e r m in u te u n d e r th e c o n d itio n s
sp ec ifie d .
The s u b s t r a t e s o lu tio n w a s 0.0.01 M HPA or HPLA in 0 .0 2 5
M Tris b u f f e r , pH 7 . 5 , c o n ta i n in g 0 . 5 M N a C l . . The c h a n g e in o p tic a l
d e n s i t y w a s r e c o r d e d on a G ilfo rd s p e c tr o p h o to m e te r .
A fu ll s c a l e
a b s o r b a n c e o f 0 .1 -or 0 . 2 a b s o r b a n c e u n its w a s - g e n e r a l l y e m p lo y e d .
The s p e c i f i c a c t i v i t y w a s d e te r m in e d a c c o r d in g to th e fo llo w in g
e q u a tio n :
s p e c i f i c a c t i v i t y = A& 254 , % , / min.
0 . 3 6 x mg e n z y m e /m l r e a c t i o n m ix tu re
w h e re 0 . 3 6 e q u a ls th e m o la r a b s o r b a n c e in d e x o f h ip p u ric a c i d w h ic h
i s form ed s t o i c h i o m e t r i c a l l y .
The a c t i v i t y c u rv e s w e re o b ta in e d b y p ip e ttin g a s u i t a b l e .
a l i q u o t o f .the e x p e r im e n ta l e n z y m e s o lu tio n in to 1,5 ml o f s u b s t r a t e
s o lu tio n in a c u v e t t e , m ix in g a n d p l a c i n g in to .th e s p e c tr o p h o to m e te r
w ith in 15 s e c o n d s .
C a r b o x y p e p ti d a s e - A y w a s o b ta in e d from W o rth in g ­
to n B io c h e m ic a l C o r p . , F r e e h o ld , N ew J e r s e y , or w a s -p re p a re d , by th e
(2 7)
m e th o d o f A n s o n .
There w a s no d e t e c t a b l e d if f e r e n c e b e tw e e n
.th e c o m m e rc ia l p r e p a r a t io n a n d th e e n z y m e p re p a re d i n . t h i s la b o r a to r y .
Stock, s o l u t i o n s w e re p r e p a r e d by d i s s o l v i n g s u i t a b l e q u a n t i t i e s of
th e c o m m e rc ia l e n zy m e in th r e e -molar sodium, chloride- s o lu tio n a f te r
w a s h in g th e c r y s t a l s s e v e r a l tim e s w ith c o ld d e io n iz e d w a t e r .
The
p r o te in c o n c e n tr a tio n w a s d e te r m in e d b y m e a s u rin g th e o p t i c a l
d e n s i t y a t 278 m a n d u s in g a n a b s o r p t i v i t y c o n s t a n t o f 6 .4 2 x IO^
lite r m o le .
O th e r c h e m i c a ls w e re o b ta in e d from th e fo llo w in g s o u r c e s :
H i p p u r y l - D L - p h e n y l l a c t i c a c i d , (HPLA), N u tr itio n a l B io c h e m ic a ls r'
H ip p u r y l- L - p h e n y la la n i n e (HPA), M an n R e s e a r c h L a b o ra to rie s ;
1 ,1 0 - p h e n a n th r o lin e (O P), J. T. B aker C h e m ic a l C o . ; C e l l e x - T (TEAE),
-4 -
B io-R ad L a b o r a to r ie s ; g - p h e n y l p r o p i o n a t e , J. T. B aker C h e m ic a l C o . ;
K o sh la n d s r e a g e n t , C y c l o c h e m ic a l C o . ; G l y c y l t y r o s i n e , Sigm a C h e m ic a l;
Rose B engal a n d m e th y le n e b l u e , F i s c h e r S c ie n tif ic C o . ; C r y s t a l v i o l e t ,
M a t h e s o n , C o le m a n , a n d Bell; T h i o p y r o n in , a g ift from D r. J. S . B e i lin ,
P o ly t e c h n ic I n s t i t u t e o f B rooklyn.
The p h o to o x id a tio n s w e re c o n d u c te d in o n e o f tw o w a y s .
(A), A
S e a rs 5.00 w a t t s l i d e p r o je c to r w a s f o c u s e d a t a d i s t a n c e o f 18 to 2 0 cm .
in to th e bo tto m o f a p l a s t i c t e s t tu b e w h ic h w a s s u p p o rte d a t a n a n g le
in a n . i c e b a t h .
All m a n ip u la tio n s o f e n z y m e s o lu tio n s c o n ta i n in g dye
w e re c o n d u c te d in a d a r k e n e d room .
(B) The s e c o n d m e th o d , w h ic h
w a s found .to be m u ch more s u c c e s s f u l , w a s a s follovys:
th e p r o je c to r
w a s d i r e c t e d a t a m irror h e ld a t a 4 5 ° a n g le to -th e p r o je c to r l e n s .
The
p r o je c to r l e n s w a s a c t u a l l y in c o n t a c t w ith th e m ir ro r. J u s t b e lo w th e
m irror a t a . 9 0 ° a n g le to th e p r o je c to r l e n s w a s a Bell a n d H o w ell f 1.6
m o v ie p r o je c to r l e n s .
The l e n s w a s p o s i t i o n e d in such, a m a n n e r a s to
a llo w th e l ig h t to p a s s th ro u g h in a c o n d e n s in g m a n n e r.
T his le n s
e n s e m b le e n a b le d th e l i g h t to b e c o n d e n s e d to a s p o t having, a d ia m e te r
of a p p r o x im a te ly 1 .5 to 2 . 0 cm .
The s a m p le s to be p h o to o x id iz e d w e re
c o n ta i n e d in s m a ll p l a s t i c c u p s w h ic h w e re i n s e r t e d in to h o le s in an
alum inum b lo c k .
The h o le s in th e alum inum b lo c k w e re la r g e en o u g h
s o t h a t th e s a m p le c o n ta i n in g c u p s c o u ld b e s h u t off from room lig h t
b y m e a n s o f ru b b e r s t o p p e r s .
The alum inum b lo c k w a s d r il le d to a llo w
c i r c u l a t i o n . o f i c e w a t e r d u rin g th e d e t e r m i n a t i o n s .
The u s e o f th is
b lo c k a llo w e d c o n s i s t e n t p o s itio n in g o f .the s a m p le in th e li g h t beam
a n d p e rm itte d , th e u s e of v e r y s h o rt e x p o s u r e t i m e s .
T h e s e tw o m eth o d s
w ill b e re f e r r e d to a s M e th o d A and. M e th o d B in fu tu re d i s c u s s i o n .
Buffer s o l u t i o n s w e re p r e p a r e d w ith th e a id o f a C o rn in g M odel
12 r e s e a r c h p H - m e t e r .
W h e n m e ta l io n c o n ta m in a tio n w a s.to . be
a v o i d e d , a l l b u ffe r s o l u t i o n s w e re e x t r a c t e d w ith a s o lu tio n o f d i t h iz o n e in c h lo ro fo rm .
The s m a ll am o u n t o f d i s s o l v e d ch lo ro fo rm w a s
re m o v e d from th e b u ffe r b y b u b b lin g p u r if ie d a i r th ro u g h th e b u ffe r a t
room te g r p e r a t u r e .
-5 -
W h e n p l a s t i c w a re or g l a s s w a re w a s u s e d to h a n d le m e ta lfre e
s o l u t i o n s , i t w a s p r e s o a k e d in a n eth an o l-E D T A b a th for s e v e r a l h o u r s ,
r i n s e d w ith e t h a n o l , a n d a i r d r i e d .
ed in s p e c i a l d i a l y s i s c h a m b e r s .
D i a l y s i s o f s o l u t i o n s w a s c o n d u c t­
T eflon i n s e r t s w e re p r e p a r e d to fit
i n s i d e th e d i a l y s i s tu b in g a t e a c h e n d .
A n e o p re n e O - r in g w a s p la c e d
a ro u n d th e d i a l y s i s tu b in g a n d te f lo n i n s e r t to o b ta in a ti g h t s e a l .
O ne te f lo n i n s e r t w a s f it te d w ith a n O - r i n g s e a l e d s to p p e r w h ic h
c o u ld b e re m o v e d for in tr o d u c tio n o r re m o v a l o f th e s a m p l e .
Stirrin g
of. th e s o l u t i o n in, th e d i a l y s i s b a g w a s. a c c o m p lis h e d , by m e a n s o f a
s m a ll te f lo n s t i r b a r .
The e n tir e a p p a r a tu s w a s th e n s u s p e n d e d in
th e d i a l y s i s b u ff e r, a n d b o th s o l u t i o n s s tir r e d w ith a m a g n e tic
stirre r.
H y d r o ly s is o f th e p r o te in m ix tu r e s w e re c a r r ie d o u t in s e a l e d
C a r iu s tu b e s for 2 0 -2 4 h o u rs a t 110° C .
F o llo w in g h y d r o ly s is a n
a l i q u o t w a s re m o v e d a n d .d r ie d o v e r KOH in v a c u u m .
The r e s i d u e w a s
th e n d i s s o l v e d in w a t e r a n d a s u i t a b l e a l i q u o t p re p a re d a n d a n a ly z e d
for a m in o -a c id c o n t e n t .
Amino acid, a n a l y s e s w e r e 'a c c o m p l i s h e d w ith
a T e c h n ic o n Amino Acid A n a ly z e r.
PROCEDURE AND RESULTS
P h o to o x id a tio n o f c a r b o x y p e p t i d a s e - A y in th e p r e s e n c e of
I ,lO - p h e n a n th r o lin e a n d m e th y le n e b l u e . This e f f e c t w a s d e te rm in e d
b y m e a s u rin g th e p e p t i d a s e a c t i v i t y o f th e e n zy m e s o lu tio n (0 .0 2 3
m g /m l) b e fo re a n d a f t e r p h o to o x id a tio n , for b o th th e a p o a n d m e t a l c o n ta i n in g e n z y m e .
The a p o e n z y m e w a s p r e p a r e d b y i n c u b a t i n g , for
up to f if te e n m i n u t e s , th e n a t i v e e n zy m e a t 0 ° , in 0.1 M Tris b u f f e r ,
-4
pH 7 . 0 , c o n ta i n in g 1 .0 M N a C l a n d 5 x 10 . M 1 , 1 0 - p h e n a n t h r o l i n e .
The m e a s u r e m e n t of p e p t i d a s e a c t i v i t y in th e a p o e n z y m e s y s te m w a s
a c c o m p lis h e d b y a d d in g a s l i g h t m o la r e x c e s s o f a 10 * M Z n C l2
s o lu tio n t o . t h e a p o e n z y m e s y s te m j u s t p rio r to th e a s s a y .
In th e
e x p e r im e n ta l s o l u t i o n s c o n ta i n in g m e th y le n e b lu e th e c o n c e n tr a tio n of
th e dye w a s 0 .0 0 2 m g /m l.
M e th o d A w a s u s e d in th e p h o to o x id a tio n .
T ab le I s u m m a riz e s th e e x p e r im e n ta l c o m p o s itio n a n d r e s u l t s o f e a c h
d e term in a tio n .
The e f f e c t o f p h o to o x id a tio n w ith m e th y le n e b lu e in th e
p r e s e n c e o f I ,! Q - p h e n a n th r o lin e on e s t e r a s e a c t i v i t y .
F iv e m ic ro ­
l i t e r s o f a 3 . 5 8 m g /m l s o lu tio n o f c a r b o x y p e p tid a s e - A y w a s d ilu te d
to 1 .0 ml w ith 0.1 M Tris b u f f e r , pH 7 . 0 , c o n ta in in g 1 .0 M N a C l .
The fin a l c o n c e n tr a tio n o f c a r b o x y p e p t i d a s e w a s 0 .0 2 3 m g /m l. .The
r e a g e n t s 1 , 1 0 -p h e n a n th r o lin e a n d m e th y le n e b l u e , w h en in c lu d e d in
_o
th e b u ff e r, w e re a t c o n c e n t r a t i o n s o f I x 10
re sp e c tiv e ly .
M a n d 0 . 0 0 2 'm g / m l
P h o to o x id a tio n w a s by M e th o d A.
The e s t e r a s e
a c t i v i t y w a s d e te r m in e d a s d e s c r i b e d in " M e th o d s " , u s in g h ip p u r y lD L - p h e n y lla c tic a c i d a s s u b s t r a t e .
T ab le II s u m m a riz e s th e e x p e r i ­
m e n ta l c o m p o s itio n a n d r e s u l t s o f e a c h d e te r m in a tio n .
The e f f e c t o f m e th y le n e b l u e - 1 , 1 0 - p h e n a n th r o lin e p h o to o x id a tio n o f c a r b o x y p e p t i d a s e - A y on e s t e r a s e a c t i v i t y . C o n s id e r a b le
c a r e w a s t a k e n to in s u r e t h a t e a c h e x p e r im e n ta l s o lu tio n o f c a r b o x y ­
p e p t i d a s e w a s h a n d le d in a n . i d e n t i c a l m a n n e r , a n d for th e s a m e p e rio d
o f tim e .
The e x p e r im e n ta l s o lu tio n s w e re p re p a re d s o t h a t th e fin a l
c o n c e n t r a t i o n s o f r e a g e n t s w e re a s fo llo w s :
c a r b o x y p e p tid a s e - A y ,
—7 —
TABLE I
P h o to o x id a tio n o f C a r b o x y p e p t i d a s e - A y i n th e P r e s e n c e
o f I ,IO - P h e n a n th r o lin e a n d M e th y le n e Blue
l--
I
A
yx I
enzym e
ml
b u ffer
ni I
0 P
5
1.0
----
0 .7 5
.0 .2 5
0 .7 5
C.23
--- -
—
0 .2 5
0 .2 5
0 .5
0 .2 5
0.2 3
0 .5
—
B lj
5
---------- ---------------C
5
H
D
Ij
I
G
I
K
5
5
I
3
I
mi
fJB
"""
ml n
h
spec. a c t.
pspfidcco
——
.....-
5 6 .6
5 .5
5
5 0 .2
-----
S
4 4 .8
0 .5
30
5
2 4 .6
0 .5
so
I
———
5 0 .2
The i n i t i a l c o n c e n t r a t i o n s o f r e a g e n t s in 0 ,1 M Tris b u ff e r, pH 7 . 0 ,
_3
c o n ta i n in g 1 .0 M N a C l w e re a s fo llo w s : MB, 0 .0 0 2 m g /m l; O P , 2 x 15
M;
Zn? + , IO- 1 M . P h o to o x id a tio n w a s b y M e th o d A, o f " M e th o d s a n d M a te ria ld '.
-8 TABLE II
The e f f e c t o f P h o to o x id a tio n w ith M e th y le n e B l u e - 1 , 10p h e n a n th r o lin e on E s t e r a s e A c tiv ity
jenzyma
A
B
I
8
I
5
I
0
F
F'
ml
OP
1.0
—
05
0 .5
—
I
5
I
5
I
I
I
ml
3
ispoe. act. cpoc. act.
!estoraoe peptidase
hV
yu.1
Znz*
—
—
7 74
565
—
——
---—
128
3 .7 2
0.5
0 .5
30
IO
0.5
——
0 .5
30
—
650
—
0.5
0.5
60
10
223
14.1
0.5
SO
—
640
37.2
6
0 I 8
:
ml
b u ff or
0
5
I
-
'
305
•
The i n i t i a l c o n c e n t r a t i o n s o f a l l r e a g e n t s in 0 .1 M Tris b u f f e r , pH 7 . 0 ,_3
c o n ta i n in g 1 .0 M N a C l w e re a s fo llo w s : MB, 0 .0 0 2 m g /m l; O P , 2 x 10
M;
Zn^+ , 10~1 M . P h o to o x id a tio n w a s b y M e th o d A.
—9 —
0 .0 2 3 m g /m l; O P , I x 10 ^ M; m e th y le n e b l u e , 0 .0 0 2 m g /m l.
All
c o m p o n e n ts w e re d i s s o l v e d in 0 . 1 'M Tris b u ff e r, pH 7 . 0 , c o n ta in in g
1 .0 M N a C l a t 0 ° C .
The e s t e r a s e a c t i v i t y w a s d e te r m in e d a s d e s c r i b e d
in. " M e th o d s " , u s in g h ip p u r y l - D L - p h e n y l l a c t i c a c id a s s u b s t r a t e .
P h o to o x id a tio n w a s d o n e u s in g M e th o d A.
T a b le III s h o w s th e e x p e r i ­
m e n ta l c o m p o s itio n a n d r e s u l t s o f e a c h d e te r m in a tio n .
P h o to o x id a tio n o f c a r b o x y p e p t i d a s e - A y w ith , in c r e a s e d
m e th y le n e b lu e a n d Rose B engal c o n c e n t r a t i o n s . In a n a tte m p t to
i n c r e a s e th e e f f i c i e n c y o f th e p h o to o x id a tio n p r o c e s s , th e c o n c e n t r a ­
tio n o f th e s e n s i t i z i n g d y e w a s i n c r e a s e d .
The p h o to o x id a tio n s tu d ie s
w e re e x p a n d e d to in c lu d e Rose B engal s e n s i t i z a t i o n a s w e ll a s t h a t o f
m e th y le n e b l u e .
The e f f e c t i v e n e s s o f Rose B engal a s a s e n s i t i z e r w a s
d e te r m in e d b o th in th e p r e s e n c e a n d a b s e n c e o f th e m e ta l c h e la to r
1 ,1 0 - p h e n a n t h r o l i n e . . The f in a l c o n c e n t r a t i o n s o f th e c o m p o n e n ts of
e a c h d e te r m in a tio n in 0 .1 M Tris b u ff e r, pH 7 . 0 , c o n ta i n in g 1 .0 M N aC l
w e re a s fo llo w s : c a r b o x y p e p tid a s e - A y , 0 .0 2 3 mg/ml;. 1, 1 0 - p h e n a n th r o _O
lin e , I x 10
M; m e th y le n e b l u e , 0 ..095 m g /m l;. Rose B e n g a l, 0 .0 9 5
m g /m l. ■ P h o to o x id a tio n w a s a c c o m p lis h e d by f o c u s in g a 500 w a tt
p r o je c to r on th e s a m p le from a d i s t a n c e o f 18-20 cm .
The e s t e r a s e
a c t i v i t y w a s m e a s u r e d a s d e s c r i b e d in " M e th o d s " , u s in g h ip p u ry l-D L p h e n y lla ctic a cid a s s u b s tr a te .
T ab le IV s u m m a riz e s th e e x p e r im e n ta l
c o m p o s itio n a n d e x p e r im e n ta l r e s u l t s o f e a c h d e te r m in a tio n .
I n h i b i t i o n ,. r e a c t i v a t i o n , a n d p h o to o x id a tio n of c a r b o x y p e p ti­
d a s e - A y in th e p r e s e n c e of EDTA. The d y e s e n s i t i z e d p h o t o l a b i l i t y of
c a r b o x y p e p tid a s e - A y w a s d e te r m in e d in th e p r e s e n c e o f a s e c o n d
m e ta l c h e l a t i n g a g e n t , EDTA. . The e x p e r im e n ta l c o n d itio n s w e re
a rra n g e d s o , t h a t th e f in a l c o n c e n t r a t i o n s o f th e c o m p o n e n ts w e re a s
fo llo w s :
c a r b o x y p e p t i d a s e - A y , 0 .0 2 3 m g /m l; EDTA, 5 x 10 ^ M;
m e th y le n e b l u e , 0 .0 0 2 m g /m l.
All c o m p o n e n ts w e re d i s s o l v e d in 0.1
M Tris b u f f e r , pH 7 . 0 , c o n ta i n in g .1.0 M N a C l a t 0° C . As in p re v io u s
e x p e r i m e n t s , r e v e r s a l o f th e in h ib iti o n re su ltin g , from th e p r e s e n c e o f
-1 0 TABLE III
The e f f e c t o f M e th y le n e B lu e - 1 , 1 0 - p h e n a n th r o lin e p h o to o x id a tio n o f
C a r b o x y p e p tid a s e - A Y o n E s t e r a s e A c tiv ity
yA l
; enzyme
A
;
U
3
ml
OP
5
ml
I
I b u ffe r
I
1.0
5
I
a5
-----
I
—
■ c
■:
5
I
I
6
d
E
I
mi
MB
I
spec. a ct.
esteraoQ
452
0 .5
|
0.5
------
------
0 .5
—
10
0.5
0 .5
-
0 .5
0 .5
....—■
"" —"
444
I
327
-----
IO o
10
374
1- —
409
IO
21 0
5
EO
I
5
I
O
I
f
&
-
§
I
min
hv
a s
Q Ii
G
I -----_ _ _ _ _ _ I!_ _ _ _ _ _ _ _ _ _ I--------------
0 .5
I
I
0 .5
,
30
_O
CP 1, 1 0 - p h e n a n th r o lin e , 2 x 10
M; MB, m e th y le n e b l u e , 0 .0 0 2 m g /m l;
Zn2+, Z nC lg , 0.1 M; w e re d i s s o l v e d in 0 .1 M Tris b u f f e r , pH 7 . 0 , c o n t a i n ­
in g 1 .0 M N a C l . All d a t a c o l l e c t e d a t 0 ° C . P h o to o x id a tio n w a s by
M e th o d A.
-1 1 TABLE IV
P h o to o x id a tio n o f C a r b o x y p e p t i d a s e -A y w ith i n c r e a s e d
M e th y le n e Blue a n d R ose B en g al C o n c e n tr a tio n s
Ij
5
I
B
,!
5
■ —*
C
I
5
——
-----
yUd
Zn^
ppoc. GCt. I
I GSfcrccc
IO
141
IO
433
10
4 9 .4
IO
374
-- -
360
-
—
0.5
-----
0.5
—
I
----
0.5
-----
30
I
O
to
to
d
I
0 .5
50
O.o
I
I
F
5
_ _ _ _ _ _ Si- - - - - - - - - - - -
0 .5
IO
d
e
i 5
I 5
0.5
O
h
I
min
I h-<r
I
A
I
I
mi
RB
(O
O
—
I
5
IO
Q
------- B
ml
OP
m3
MB
O
SO
I /U
I m!
1snzym e I b u ffer
4 2.3
The c o m p o n e n ts : MB, m e th y le n e b l u e , 0 . 0 0 2 m g /m l: O P, 1,10 p h e n a n th r o
l i n e , 2 x IO"3 M; RB, R ose B e n g a l, 0 .0 9 5 m g /m l; Znz + , Z n C l 2 , 0.1 M; w e re
d i s s o l v e d in 0 .1 M T ris b u f f e r , pH 7 . 0 , c o n ta i n in g 1.0 M N a C l . All d a t a
o b ta in e d a t 0 ° C . P h o to o x id a tio n w a s b y M e th o d A.
I
-1 2 -
th e m e ta l c h e l a t o r w a s a c c o m p l i s h e d b y th e a d d itio n o f a p p ro p ria te
q u a n t i t i e s o f 10 1 M Z n C l2 in 0.1 M Tris b u ff e r, pH 7 . 0 , c o n ta in in g
1 .0 M N a C l .
The p e p t i d a s e a c t i v i t y w a s d e te r m in e d a s d e s c r i b e d in
" M e th o d s " , u s i n g h ip p u r y l - L - p h e n y l a l a n i n e a s s u b s t r a t e .
T a b le V
s u m m a riz e s th e e x p e r im e n ta l c o m p o s itio n a n d r e s u l t s o f e a c h d e te r m i­
n a tio n .
. P h o t o s e n s i t i z a t i o n w ith 1 , 1 0 - p h e n a n t h r o l i n e .
The p h o t o s e n s i ­
ti z i n g e f f e c t o f 1,1 0 - p h e n a n th r o lin e w a s d e te r m in e d in. th e fo llo w in g
m anner:
c a r b o x y p e p t i d a s e - A y w a s d i s s o l v e d in 0.1 M Tris b u ff e r, pH
7 . 0 , c o n ta i n in g 1.0. M N a C l a t 0 ° C . , a t a c o n c e n tr a tio n o f 0.6 1 m g /m l.
M e th y le n e b lu e a n d . 1 , 1 0 - p h e n a n th r o lin e , w h e n u s e d , w e re d i s s o l v e d
. i n 0.1 M Tris b u f f e r , p H . 7.0/ c o n ta i n in g 1 .0 M N aC l a t c o n c e n tr a tio n s
o f 0 .0 9 9 mg/ml. a n d I x 10
M re sp ec tiv e ly .
Ten m ic r o lit e r a liq u o ts
o f a 0.1 M Z n C l2 s o lu tio n w e re u s e d to r e v e r s e th e e f f e c t s o f 1 , 10p h e n a n t h r o l i n e . P e p t i d a s e a c t i v i t y w a s d e te rm in e d a s d e s c r i b e d in
" M e th o d s " , u s in g h ip p u r y l - L - p h e n y l a l a n i n e a s s u b s t r a t e .
o x id a tio n w a s b y /M e th o d A.
P h o to ­
T ab le VI s u m m a riz e s th e e x p e r im e n ta l
c o m p o s itio n a n d r e s u l t s o f . t h e s e d e t e r m i n a t i o n s .
P r e p a r a tio n o f a p o e n z y m e on S e p h a d e x G - 2 5 c o lu m n .
A p o c ar(3)
b o x y p e p tid a s e - A y w a s p r e p a r e d a c c o r d in g to th e m e th o d s o f V a lle e C '
A 10 ml h y p o d e rm ic s y r in g e w a s prep ared , a s a c h ro m a to g ra p h y co lu m n .
■A s m a ll f i l t e r p a p e r d i s c w a s c u t a n d .p la c e d , in. th e e n d o f th e sy rin g e
to s e r v e a s a. s u p p o rt for th e S e p h a d e x r e s i n .
The f i l t e r p a p e r s u p p o rt
w a s w a s h e d w ith 6 N H C l , a n d w ith d e i o n i z e d w a t e r .
The S e p h a d e x
G - 2 5-r e s i n w a s th e n s u s p e n d e d in 0.1 M Tris b u ffe r, pH 7 . 5 , c o n t a i n ­
i n g 1 .0 M N a C l , a n d p l a c e d u n d e r a v a c u u m to .re m o v e a i r .
This
s u s p e n s i o n .then w a s p o u re d in to th e s y r in g e c o m p le tin g th e p r e p a r a tio n
o f th e c o lu m n .
The co lu m n w a s fitte d , w ith .a c o n s t a n t p r e s s u r e r e s e r ­
v o ir . . The c a p a c i t y o f;th e colum n to r e t a i n 1 , 1 0 - p h e n a n th r o lin e w a s
d e te r m in e d by p l a c i n g 1 .0 ml of a 0.1 M Tris b u ff e r, pH 7 . 5 , c o n ta in in g
1 .0/ M N a C l , s a t u r a t e d With 1, 1 0 - p h e n a n th r o lin e on th e co lu m n and
-1 3 TABLE V
I n h ib i tio n , R e a c t i v a t i o n , a n d P h o to o x id a tio n o f
C a r b o x y p e p ti d a s e - A y in th e p r e s e n c e o f EDTA
I
/A l
enzym a
mI
b u ffe r
ml
EDTA
A
jj
5
1.0
B
I
i'
5
0.75
0 .2 5
0.75
0 .2 5
0 .2 5
0 .2 5
c
ml
M3
—
I
min
hv
I
—
i
— I —
s p e c . co t.
p ep tid a se
&
5
I
45.2
5
542
5
22.6
5
D
s
----------J -----------------
0 .5
30
The i n i t i a l c o n c e n t r a t i o n s o f r e a g e n t s in 0 .1 M Tris b u ff e r, pH 7 . 0 , c o n t a i n ­
in g 1.0 M N a C l w e re a s fo llo w s : EDTA, 2 x 10- 3 M; MB, 0 . 0 0 4 m g /m l;
Z n C l2 - 0 .1 M . P h o to o x id a tio n M e th o d A.
-1 4 TABLE VI
P h o t o s e n s i t i z a t i o n o f 1,1 0 - P h e n a n th r o li n e
I
! onzvm e
mI
o P
ml
MB
ml
b u ffe r
A
100
0 .5
———
0 .5
B
ICO
0 .5
C
100
0 .5
D
100
E
ICO
min
h?r
cpec. a c t.
pcptldaso
.
———
17. 7
0 .3
—
10
6 8 .3
—
0 .3
30
10
GGC
---—
........
1.0
.....—
... —
755
—
0 .5
0 .5
30
■■■ —
5 7 .4
The i n i t i a l c o n c e n t r a t i o n s o f r e a g e n t s d i s s o l v e d in 0 .1 M Tris b u ff e r, pH
7 . 0 , c o n ta i n in g 1 .0 M N a C l w e re a s fo llo w s : O P , 2 x IO- ^ M; MB, 0 .018
m g /m l; ZnClg , 0.1 M . P h o to o x id a tio n M e th o d A.
I
-1 5 e l u tin g w ith 0.1 M T r i s , 1 .0 M N a C l , pH 7 . 5 b u ffe r,
th e 1 , 1 0 - p h e n a n -
th ro lin e d id n o t a p p e a r u n til a t l e a s t 2 00. d ro p s of e l u e n t h a d b e e n
c o lle cte d .
The a c t u a l p r e p a r a t io n o f th e a p o e n z y m e w a s a s fo llo w s :
10 ijl
o f a c a r b o x y p e p t i d a s e - A y s o l u t i o n ( 4 .0 3 m g /m l) w a s i n c u b a t e d for one
h our a t 0° C . w ith I ml o f 0.1 M Tris b u f f e r , pH 7 . 5 , c o n ta i n in g 1.0- M
N a C l a n d s a t u r a t e d w ith 1 , 1 0 - p h e n a n t h r o l i n e .
The s o lu tio n - th e n w a s
p l a c e d on th e colum n d e s c r i b e d a b o v e a n d e l u te d w ith 0.1 M Tris
b u f f e r , pH 7 . 5 a t 0 ° C . , c o n ta in in g 1.0 M N a C l .
The f r a c tio n c o n t a i n ­
in g d ro p s 78 to 12 3 w a s c o l l e c t e d in a p l a s t i c tu b e w h ic h h a d b e e n
c a r e f u l ly t r e a t e d w ith a 95% eth an o l-E D T A s o lu tio n , to- r e d u c e m e ta l io n
c o n ta m in a ti o n . . This e n z y m e - c o n t a i n i n g fr a c tio n th e n w a s a s s a y e d for
p e p tid a se a c tiv ity .
The p r e s e n c e o f a p o e n z y m e w a s d e m o n s t r a t e d by
th e a b i l i t y to i n c r e a s e th e p e p t i d a s e a c t i v i t y 60% upon th e a d d itio n
2+
o f a s m a ll q u a n tity o f Zn
a s sh o w n in T ab le V II.
P r e c a u tio n s w e re t a k e n a g a i n s t m e ta l ion c o n ta m in a tio n to
o b ta in s a t i s f a c t o r y r e s u l t s w h e n th e a p o e n z y m e s o lu tio n w a s a s s a y e d .
All c u v e t t e s a n d p i p e t t e s w e re t r e a t e d b e fo re h a n d in an ^ O - E t h a n o l
( 5 0 : 5 0 , v:v) b a th c o n ta i n in g EDTA. F o llo w in g t h i s s o a k i n g , t h e y w ere
r i n s e d w ith d e i o n i z e d w a t e r .
The s u b s t r a t e a n d a ll b u ffe rs w h ic h cam e
in c o n t a c t w ith th e a p o e n z y m e w e re e x t r a c t e d w ith d ith iz o n e b efo re
•use.
O n ly u n d e r t h e s e c o n d itio n s c o u ld r e l i a b l e a s s a y s b e o b ta in e d .
P h o to o x id a tio n of. a p 'o c a r b o x y p e p t id a s e - A y w ith m e th y le n e b lu e .
A p o c a r b o x y p e p tid a s e - A y w a s p re p a re d a s p r e v io u s ly d e s c r i b e d , and
a s s a y e d for re m a in in g a n d r e a c t i v a t i b l e p e p t i d a s e a c t i v i t y .
The
r e s u l t s o f t h e s e a s s a y s a re g iv e n in T a b le VIII.
The p h o t o l a b i l i t y of t h i s a p o e n z y m e p r e p a r a tio n w a s th e n
d e te r m in e d in th e fo llo w in g b u ffe r s o lu tio n ; 0.1 M Tris , I. O M N a C l ,
pH 7 . 5 a t 0 ° C . b u ffe r a n d 0.1 M Tris b u f f e r , pH 7 . 5 a t 0 ° C . , c o n ­
t a in in g 1 .0 M N a C l arid. 0.199 m g /m l m e th y le n e b l u e , a n d an i d e n t i c a l
m e th y le n e b lu e fre e b u ffe r e x t r a c t e d w ith a. c a r b o n te tr a c h lo r i d e d ith iz o n e
-1 6 TABLE V II
P r e p a r a tio n o f A p o enzym e
on S e p h a d e x G -2 5 Colum n
yu.1
opoenzym e
yuul
Z
100
------
100
10
A A z ^m In 2 5 4 m /t
n ~ r
0 .0 0 G 4
i
0 .0 1 5 6
The Z n C l9 , O.1 M , w a s d i s s o l v e d in 0 .1 M Tris b u ff e r, pH 7 . 0 , c o n ta i n in g
1 .0 M N a t l .
‘IOe N N O'T
Buiuteiuoo ' o ' Z Hd 'Jajjn q s p i IAI I ' O uT 1A[ VO SBM uonejiueouoD ZiQuz
OOI
I
OSI OO
OOI
COOO O
-Viii tpSH u 1uiy-V 17
\
,^U z
)rZ
oiuAzuood 0
suoi OUTZ qq.TM
^ v - 9SepTidadAxoqjeoodv jo UOTjeAnoeay
IIIA 318VI
-ZT-
-1 8 -
s o lu tio n to rem o v e c o n ta m in a tin g m e ta l i o n s . ' The tr a c e q u a n t i t i e s of
c a r b o n te tr a c h lo r i d e d i s s o l v e d in t h e s e b u ff e rs w e re re m o v e d b y b u b b lin g
a i r w h ic h h a d b e e n p u rif ie d b y p a s s i n g th ro u g h s u lf u r ic a c i d a n d c a lc iu m
h y d ro x id e s o l u t i o n s th ro u g h t h e m .
The b u ffe rs w e re th e n u s e d to p r e ­
p a re th e s o l u t i o n s d e s c r i b e d in T ab le .IX.
P r e c a u tio n s w e re ta k e n
a g a i n s t m e ta l io n c o n ta m in a tio n w h e re n e c e s s a r y a s p r e v io u s ly
d e sc rib e d .
P h o to o x id a tio n o f c a r b o x y p e p t i d a s e - A y a n d peptjda&e; a c t i v i t y .
The e f f e c t o f p h o to o x id a tio n on th e p e p t i d a s e a c t i v i t y o f c a r b o x y p e p tid a s e - A y w a s d e te r m in e d in th e p r e s e n c e o f R o se B engal a s w e ll a s the
m e th y le n e b lu e 1 , 1 0 -p h e n a n th r o lin e s y s t e m .
The fin a l c o n c e n tr a tio n s
o f a l l r e a g e n t s in 0.1 M Tris b u ff e r, pH 7 . 0 a t 0°. C . , c o n ta i n in g 1 .0 M
N a C l w e re a s fo llo w s :
c a r b o x y p e p tid a s e - A y , 0 .0 2 3 m g /m l, 1 , 1 0 - p h e n a n ­
t h r o l i n e , I x 10 3 M , m e th y le n e b l u e , 0 .0 9 9 m g /m l, Rose B e n g a l, 0 .0 9 5
m g /m l.
The te c h n i q u e u s e d in th e p h o to o x id a tio n w a s t h a t of M eth o d A.
The p e p t i d a s e a c t i v i t y w a s d e te r m in e d a s d e s c r i b e d in " M e th o d s " ,'
u s in g h i p p u r y l - L - p h e n y l a l a n i n e a s s u b s t r a t e .
As in. p r e v io u s c a s e s , 1 ,1 0 - p h e n a n th r o lin e in h ib iti o n w a s
re v e rs e d , b y th e a d d itio n o f a s u i t a b l e a l i q u o t o f 0.1 M ZnClg s o lu tio n
j u s t p rio r to. a s s a y .
T ab le X s u m m a riz e s th e e x p e r im e n ta l c o m p o s itio n
a n d r e s u l t s o f e a c h d e te r m in a tio n .
R ate o f l o s s o f p e p t i d a s e a c t i v i t y o f c a r b o x y p e p t i d a s e - A y w hen
p h o to o x id iz e d in p r e s e n c e o f R ose B e n g a l.
Ten m ic r o lit e r s o f a
c a r b o x y p e p tid a s e - A y s o lu tio n ( 2 . 8 m g /m l) w a s d ilu te d to. 1 .0 ml w ith
0 .1 M Tris b u ff e r, pH 7 , 5 a t 4°. C . , c o n ta i n in g 1.0, M N a C l a n d 0 .0 4 9
m g /m l R ose B e n g a l, g iv in g a f in a l e n z y m e c o n c e n tr a tio n o f 0 .0 2 8 m g /m l.
This s o lu tio n w a s t h e n e x p o s e d to s e v e r a l in te r v a ls o f l i g h t , s u b s e q u e n t
to w h ic h 50' p.l w e re re m o v e d a n d a n a l y z e d for p e p t i d a s e a c t i v i t y .
P h o to o x id a tio n w a s d o n e u s in g M e th o d B . P e p t i d a s e a c t i v i t y w a s d e ­
te rm in e d a s d e s c r i b e d in " M e th o d s " , u s in g h i p p u r y l - L - p h e n y l a l a n i n e
as su b stra te .
The d a t a in T a b le XI s u m m a riz e th e r e s u l t s o f t h e s e
-19 —
TABLE IX
P h o to o x id a tio n o f A p o c a r b o x y p e p tid a s e - A y
w ith M e th y l e n e Blue
yA.8
5 ml i
apocnz. I buffer I
ml
M3
0.3
D
I
Zr^
-----
100
I 0.3
I
I
100
i
?
0.3
0.5
_________ I _______ I__________
30
!
1.5
---- 1.5
|AA>
!r2 5 4
^ 0 .0052
I
I 0.0070
0.0074
& 0.0162
The i n i t i a l c o n c e n t r a t i o n s o f r e a g e n t s d i s s o l v e d in 0 .1 M Tris
b u f f e r , _pH 7 . 0 , c o n ta i n in g 1 .0 M N a C l w e re a s fo llo w s : O P,
2 x 1 0 "^ M; MB , 0.199 m g /m l; ZnClg , 0.1 M . a A /m in 254 mu
a re r e p o r te d h e re b e c a u s e a p o e n z y m e c o n c e n tr a tio n w a s n o t
d e te r m in e d . P h o to o x id a tio n M e th o d A.
I
—2 OTABLE X
P h o to o x id a tio n o f C a r b o x y p e p tid a s e - A y
a n d P e p tid a s e A c tiv ity
yx\
I ml
enzym e! b u ffer
A
5
I
ml
MB
mi
OP
ml
RB
j
i
min
b tr
spec. act.
peptidase
yM-l
5 0 .4
1.0
B
.
O
'
D
I
e
'
i
5
—
0 .5
Cl
■
—
0.5
5
5
—
OS
----0 .5
________ L - . ~
------
0 .5
0.5
----
.
5
0.5
0
i|
5
0 .5
0.5
_
—
—
0 .5
-----
F
H
S
I______ U________
j
I
----
j 30
I
-----
0.5
' 30
I________ ________
I
05
10
5.7
10
42.1
10
—
IO
!9.5
-----
30
----
j
51.3
—
0.5
30
-----
I
43
—
0 .5
—
-----
j
4 4 .2
j
The i n i t i a l c o n c e n t r a t i o n s o f r e a g e n t s in 0 .1 M Tris b u f f e r , pH 7 . 0 ,
c o n ta i n in g 1 .0 M N a C l w e re a s fo llo w s : MB, 0.199 m g /m l; RB, 0.198
m g /m l; O P , 2 x IO-3 M; ZnClg , 0 .1 M . P h o to o x id a tio n M e th o d A.
-2 1 TABLE XI
R ate o f l o s s o f P e p t i d a s e A c tiv ity o f C a r b o x y p e p ti d a s e - A y
w h e n P h o to o x id iz e d in p r e s e n c e o f R ose B engal
min
h i/-
O
AAy/ m!n 2 5 4 nyx
I
0 .0 2 0 0
I
2
i
I
act. pcptidaso
61.5
0 .0 1 7 4
I
53 .5
0 .0 0 CO
I
2 4 .6
|
0 .0 0 7 2
21.5
0 .0 0 5 2
15.4
0 .0 0 4 8
14.3
0 .0 0 3 2
9 .3
0 .0 0 3 4
10.4
"
0 .0 0 2 3
8.6
12
0 .0 0 3 0
9 .2
3
I
I
sp ec.
5
-
7
...............
9
.
,
13
I
,s
I
0 .0 0 2 3
0 .0
0 .0 0 3 4
10 . 4
.
'
' -
-2 2 -
d e te r m in a tio n s .
The d a ta o f T ab le XII s u m m a riz e .the r e s u l t s o f an
i d e n t i c a l e x p e r im e n t in w h ic h e s t e r a s e a c t i v i t y w a s d e te r m in e d .
R ose B engal p h o t o o x i d a t i o n , e s t e r a s e a c t i v i t y .
Ten m ic r o lite r s
o f a c a r b o x y p e p t i d a s e s o lu tio n ( 2 . 8 m g /m l) in Tris b u ffe r w a s d ilu te d to
1 .0 ml w ith 0 . 1 M Tris b u f f e r , pH 7 . 5 a t 4 ° C . , c o n ta i n in g 1 .0 M N a C l
a n d 0.049- m g /m l Rose B e n g a l.
This s o lu tio n th e n w a s d iv id e d am ong
s e v e n s m a ll p l a s t i c c u p s w h ic h w e re h e ld in a n alum inum b l o c k . The
b lo c k w a s c o o le d by a llo w in g i t to s t a n d in a n i c e b a t h .
E ach p l a s t i c
cup w a s c a p a b l e o f b e in g i n d e p e n d e n tly c o v e r e d in s u c h a m a n n e r a s
to e x c lu d e a l l l i g h t .
F o llo w in g th e d is t r i b u t i o n of th e en zy m e s o l u ­
t i o n , th e li g h t s o u r c e w a s tu rn e d on a n d e a c h cup c o v e r e d a f t e r a
c e r t a i n e x p o s u r e . t i m e . The d a t a in T ab le XIII s u m m a riz e th e r e s u l t s
of t h i s d e te r m in a tio n .
The e s t e r a s e a c t i v i t y w a s d e te r m in e d a s d e ­
s c r i b e d in " M e th o d s " , u s in g h ip p u r y l - D L - p h e n y l l a c t i c a c i d a s
su b strate .
The d a ta in T a b le s XIV a n d XV s u m m a riz e th e r e s u l t s of
sim ila r d e term in a tio n s.
The lig h t s o u r c e em p lo y e d w a s t h a t d e s c r ib e d
in M e th o d B o f " M e th o d s a n d M a t e r i a l s " .
The d a ta o f T ab le XVI
s u m m a riz e an i d e n t i c a l e x p e r im e n t in w h ic h p e p t i d a s e a c t i v i t y w a s
m easured.
P h o to o x id a tio n o f c a r b o x y p e p t i d a s e -A v w ith c r y s t a l v i o l e t
s e n s i t i z e r . A c r y s t a l v i o l e t s o lu tio n w a s p re p a re d b y d i s s o l v i n g 3 .9
mg o f c r y s t a l v i o l e t d y e in 38 ml of 0.1 M Tris b u ff e r, pH 7 . 5 a t 2 5°
C . , c o n ta i n in g 1 .0 M N a C l . J i v e 'm i c r o l i t e r s of a c a r b o x y p e p tid a s e - A y
s o lu tio n . (9 .2 1 m g /m l) w a s th e n d ilu te d in th e d ark w ith 0 .2 5 ml of a 2
x 10 3 M I , iO - p h e n a n th r o lin e s o lu tio n in 0 . 1 M Tris , I . O M N a C l b u ffer
a n d w ith 0 . 2 5 ml o f th e c r y s t a l v i o l e t b u ffe r d e s c r i b e d a b o v e .
The
l i g h t s o u r c e u s e d in th e p h o to o x id a tio n w a s t h a t d e s c r i b e d in M eth o d B .
The p e p t i d a s e a c t i v i t y w a s d e te r m in e d b y th e u s u a l m e th o d s fo llo w in g
th e r e v e r s a l o f th e in h ib iti o n c a u s e d by th e 1 , 1 0 -p h e n a n th ro lin e w ith
2+
th e a d d itio n of 10 ul o f a Zn
s o l u t i o n . T ab le XVII s u m m a riz e s th e
r e s u l t s o f t h e s e d e te r m in a tio n s .
—2 3 —
TABLE XII
Rate o f l o s s o f E s t e r a s e A c tiv ity o f C a r b o x y p e p ti d a s e - A y
w h e n p h o to o x id iz e d in p r e s e n c e o f Rose B engal
time
rain
15
S
AA
SQC
3 0 sac
m i n 2 5 4 nyx
..
2
Gcti e c l e r a s o
329
0 .0 5 0 6
m o
I
169
.
I
3
epee.
0 .1 0 6 8
0 .0 5 5 0
1
?
. .
I
0 .0 2 5 0
____
. ___
0 .0 2 2 3
77
__ ' ........|
70
0 .0 2 1 8
33
I
0 .0 2 4 8
74
rzzzz
0 .0 1 9 2
59
0 .0 1 8 2
56
4
.
5
I
I
!
.
-24-
TABLE XIII
R o se B en g al P h o t o o x i d a t i o n , E s t e r a s e A c tiv ity
30 0
A k y rmin
2 5 4 rcy*.
o p oc.
OC L octoroso
0 . 1 2 1S
382
0.0952
277
a
timo
0 .0 7 8 8
242
CO
0 .0 7 7 6
239
120
0.0 0 6 0
174
.......................
I
180
0 .0 3 0 8
II9
j
560
0 .0 2 3 0
71
-25TABLE XIV
R ose B engal P h o to o x id a tio n , E s te ra s e A c tiv ity
tim e
so c
6 A / min 2 5 4 m&q.
spec. act. eotorcss
- - ....................... -
-----------
- .......-
0.0920
283
°
15
0.0760
234
30
0.0634
204
45
0.0660
205
GO
0.0548
16 9
75
0 .0 5 1 6
158
90
0.0432
148
105
0.0332
102
120
0.0342
105
420
0 .0 1 1 2
34J5
-
•
-
—
26—
TABLE XV
■Rose B e n g a l P h o t o o x i d a t i o n , E s t e r a s e A c t i v i t y
tim o
coc
|
0 .0 8 8 0
;
s p o c . a c t . e st er ase
271
155
30
0.0555
HO
43
0.023G
73
SO
0 .0212
65
15
\
73
0.0176
I
54
so
0 .0 1 4 4
44
IOS
0.01 5 3
42
O
CJ
i
|
0 .0 5 0 4
I
I
A A / min 2 5 4 r y u *
0 .0 1 0 4
32
I
-27-
TABLE XVI
R ose B engal P h o t o o x i d a t i o n , P e p t i d a s e A c tiv ity
ti m e
in f sec
I
I
I
A A y Z min 2 5 4 m / * .
spec.
0 .0 1 4 6
|
0 .0 0 3 0
I
cct. peptidase
45.0
24.5
SO
0 .0 0 5 2
16.0
I
45
0 .0 0 4 2
13.0
!
60
0 .0 0 3 2
75
I
90
I
0 .0 0 3 2
.............. ...... ...............
0 .0 0 3 0
3.8
9.8
-----
9.0
-2 8 TABLE XVII
P h o to o x id a tio n o f C a rb o x y p e p tid a s e -A y w ith
C r y s ta l V io let S e n s itiz e r
I
e nz y me
'
.1
A
5
I_____ _____
I
B
I
5
I
c
I
5
0P
I
I
b u ffer
jil
I m ,n
spoc. act.
peptidase
Z n z*
I
67.4
0.25
|
0.25
——
—-
:
10.0
0.25
g
0.25 ' '
5
i
I___________ I
10
!
Gl-S
I____________
J
IO
I
5
0.25
0.25
I F
I
5
:l
0.25
I---------- 5----------------- 3-----------------
I
10
65.9
S
O
D
S
d
10
l£>
—
LU
0.25
J
0.25
_________ ______
,3
10
63 .3
0 .2 5
50
10
573
The i n i t i a l c o n c e n t r a t i o n s o f r e a g e n t s , d i s s o l v e d in 0.1 M Tris b u ffe r,
pH 7 . 0 , c o n ta i n in g 1 .0 M N a C l w e re a s fo llo w s : O P , 2 x 10“ ^ M;
C V , 1. 02 m g /m l; Z n C lg , o . l M . P h o to o x id a tio n w a s by M e th o d B.
-29-
pH D e p e n d e n c e of p h o to o x id a tio n o f C a r b o x y p e p ti d a s e - A y in th e
p r e s e n c e o f I ,I Q - p h e n a n th r o lin e a n d m e th y le n e b l u e .
Tw enty m ic r o lite r s
o f a 2 x 10 ^ M 1 ,1 0 - p h e n a n th r o lin e s o lu tio n in 0.1 M Tris b u ff e r, c o n ­
ta i n i n g 1 .0 M N a C l , a n d 2 0 ml o f a 0.198 m g /m l so lu tio n , o f m e th y le n e
b lu e in th e s a m e b u ffe r w e re m ix ed a n d th e pH a d j u s t e d to th e fo llo w in g
v a lu e s a t 0° C .:
8 ..88, 8 .4 7 ., 7 . 9 6 , 7 . 5 5 , 6 . 9 6 , 6 . 5 2 , 5 . 4 9 .
As e a c h
p H v a lu e w a s o b t a i n e d , tw o I ml a l i q u o t s w e re r e m o v e d , e a c h a liq u o t
p l a c e d in a s e p a r a t e t u b e , a n d k e p t a t 0 ° C .
The p a i r of a l i q u o t s , for
e a c h i n d i c a t e d pH v a l u e , w e re th e n t r e a t e d in th e fo llo w in g m anner:
fiv e m ic r o lit e r s o f a s o lu tio n o f c a r b o x y p e p tid a s e - A y ( 4 . 0 3 m g /m l) w ere
in tr o d u c e d in to e a c h t u b e .
O ne tu b e w a s k e p t in th e d a r k , w h ile th e o th e r
w a s p h o to o x id iz e d for t e n m i n u t e s .
A fter t h i s te n m in u te p e r io d , b o th
tu b e s w e re t r e a t e d w ith 10 u I o f a 10 1 M Z n C l2 s o l u t i o n , a n d th e p e p t i ­
d a s e a c t i v i t y d e te r m in e d . .P h o to o x id a tio n w a s a c c o m p lis h e d by f o c u s in g
a 500 w a t t p r o je c to r on th e s a m p le from a d i s t a n c e o f 18-20 c m .
p e p t i d a s e a c t i v i t y w a s d e te r m in e d a s p r e v i o u s l y d e s c r i b e d .
The
Table XVIII
s u m m a riz e s th e r e s u l t s of t h e s e d e t e r m i n a t i o n s .
pH D e p e n d e n c e o f p h o to o x id a tio n o f c a r b o x y p e p t i d a s e - A y w ith
R ose B engal a s s e n s i t i z e r .
Tris b u f f e r , 0.1 M , c o n ta in in g 1 .0 M N aC l
w a s u s e d to p ro d u c e b u ffe rs h a v in g th e pH v a l u e s a t 0° a s sh o w n in
T ab le XIX.
C o n c e n tr a te d H C l w a s a d d e d to th e s ta r tin g s o lu tio n u n til
e a c h pH v a lu e w a s a t t a i n e d .
D uring t h i s p r o c e d u r e , 0 . 5 ml o f e a c h
s u c c e e d i n g b u ffe r w a s re m o v e d a n d p l a c e d in a t e s t tu b e .
.
R ose B engal
( 0. 1 m g /m l) d i s s o l v e d in 0.1 M Tris b u ff e r, c o n ta in in g 1.0 M N a C l , w a s
l i k e w i s e a d j u s t e d to th e i n d i c a t e d pH v a l u e s a t 0° C . A gain a s e a c h pH
v a lu e w a s a t t a i n e d , 0 . 5 ml w a s re m o v e d a n d m ix ed w ith i t s c o u n te r p a r t
above.
Five m ic r o lit e r s o f a c a r b o x y p e p tid a s e - A y s o lu tio n ( 3 . 5 3 m g/m l)
w a s th e n a d d e d in a d a r k e n e d room to e a c h tu b e , g iv in g a f in a l en zy m e
c o n c e n tr a tio n o f 0 . 0 2 3 m g /m l.
O ne h u n d re d m ic r o lite r s o f e a c h r e s u l t ­
ing en zy m e s o lu tio n w a s rem o v ed a n d a s s a y e d for p e p t i d a s e a c t i v i t y .
F o llo w in g t h i s i n i t i a l a c t i v i t y d e te r m in a tio n , e a c h s o lu tio n w a s e x p o s e d
for fiv e m in u te s to a 500 w a t t lig h t s o u r c e , a c c o r d in g to M e th o d A.
-3 0 TABLE XVIII
pH D e p e n d e n c e o f P h o to o x id a tio n o f C a r b o x y p e p ti d a s e - A y in
th e p r e s e n c e o f I ,I O - P h e n a n th r o lin e a n d M e th y l e n e Blue
pH
specific activity
u n cx id iz cd
specific
activity
o x id i z e d
8.83
67.4
30.1
0.47
6 5.5
31.0
7 96
60.5
32.0
7.55
-------
38.8
6 .9 6
50.5
6.52
6 7 .4
53.6
5.49
—
5 6 .5
-3 1 -
S u b s e q u e n t to th is e x p o s u r e a n o th e r o n e h u n d re d m ic r o lit e r a l i q u o t w a s
r e m o v e d , a n d th e p e p t i d a s e a c t i v i t y r e d e t e r m i n e d .
The r e s u l t s o f t h e s e
d e te r m in a tio n s a re r e p r e s e n t e d in T ab le XIX.
. E ffe ct o f p e p t i d a s e s u b s t r a t e on r a t e o f p h o to o x id a tio n w ith Rose
B e n g a l. In o rd e r to d e te r m in e i f th e p h o to c h e m ic a l d e a c t i v a t i o n of
c a r b o x y p e p tid a s e - A y w a s th e r e s u l t o f a n o x id a tio n n e a r o r a t th e a c ti v e
c e n t e r o f th e e n zy m e , a s u b s t r a t e p r o te c tio n e x p e r im e n t w a s u n d e r t a k e n .
All r e a g e n t s u s e d in th e e x p e r im e n t w e re d i s s o l v e d in 0.1 M Tris b u ffe r,
pH 7 . 0 a t 0 ° ' C. , c o n ta in in g 1 .0 M N a C l , a t th e fo llo w in g c o n c e n t r a ­
tio n s :
c a r b o x y p e p tid a .s e -A y; 0 . 0 2 3 m g /m l; Rose B en g a l, 0 . 0 9 5 m g /m l;
-4
h ip p u ry l-L -p h e n y la la n in e , 5 x 1 0
M. .P h o to o x id a tio n w a s done
a c c o r d in g to M e th o d A.
The p e p t i d a s e a c t i v i t y w a s d e te r m in e d a s
d e s c r i b e d in " M e th o d s " , u s in g h i p p u r y l - L - p h e n y l a l a n i n e a s s u b s t r a t e .
The p r e s e n c e o f p a r t i a l l y h y d ro ly z e d s u b s t r a t e d id n o t a f f e c t th e a s s a y
re su lts.
T ab le XX s u m m a riz e s th e e x p e r im e n ta l c o m p o s itio n a n d r e s u l t s
o f e a c h d e te r m in a tio n .
S u b s tr a te p r o te c tio n in th e Rose B engal s y s t e m .
S u b s tr a te
p r o te c tio n w a s a tte m p te d b y p h o to o x id iz in g c a r b o x y p e p tid a s e - A y in
-4
th e p r e s e n c e o f 5 x 10
M h i p p u r y l - L - p h e n y l a l a n i n e . The r a t e s of
d e a c t i v a t i o n in th e p r e s e n c e a n d a b s e n c e o f s u b s t r a t e w e re th e n
c o m p a re d b y p re p a rin g tw o e x p e r im e n ta l m i x t u r e s . The f in a l c o n c e n ­
t r a t i o n s o f r e a g e n t s in t h e s e m ix tu re s w e re a s fo llo w s:
c a r b o x y p e p tid a s e
Ay, 0 . 0 2 3 m g /m l; R ose B e n g a l, 0 . 0 9 5 m g /m l; a n d h ip p u r y l - L - p h e n y l
a lan in e 5 x 1 0
^M.
All r e a g e n t s w e re d i s s o l v e d in 0 . 1 . M Tris b u ffe r,
pH 7 . 0 a t 0 ° C . , c o n ta i n in g 1.0 M N a C l .
T h e s e e x p e r im e n ta l m ix tu re s
w e re th e n e x p o s e d to th r e e c o n s e c u t i v e fiv e m in u te i n t e r v a l s o f lig h t.
B etw een e a c h i n te r v a l a s u i t a b l e a l i q u o t w a s rem o v ed a n d th e p e p t i d a s e
a c t i v i t y d e te r m in e d in th e u s u a l m a n n e r. .M ethod A w a s u s e d to p h o to o x id iz e th e e n z y m e .
T ab le XXI s u m m a riz e s th e e x p e r im e n ta l c o m p o s itio n
a n d r e s u l t s o f t h e s e d e te r m i n a t i o n s .
-32-
TABLE XIX
pH D e p e n d e n c e o f P h o to o x id a tio n o f C a r b o x y p e p ti d a s e - A y
w ith R ose B engal a s s e n s i t i z e r
pH
c p e c . act. unoxidized
s p e c . act. o x id i z e d
9.0
30.3
2.37
3.5
30.0
1 .8 9
8.0
30.3
3.31
7 6
30.8
4.73
,0
30.8
6.15
6.5
27 .9
7.10
6.0
30.8
7.56
-3 3 -
TABLE XX
E ffe c t o f P e p t i d a s e S u b s tr a te on Rate o f
P h o to o x id a tio n w ith R o se B engal
enzyme
ml
b u f f or
y
*1
ml
su b.
in I
RB
min
htr
A
I
5
0 .5
, -----
0.5
3
S
5
0.5
-----
0 .5
50
5
0.5
0.5
-----
—
0 .5
50
C
D
I
5
0.5
I
spec. act.
peptidase
3 9 .0
3.6
45.4
645
-3 4 TABLE XXI
S u b s t r a t e P r o t e c t i o n , R ose B engal S y stem
yU I
v enzymo
|
ml
!
s s ub st ra te I
I
i
0.5
A
B
6
I
5
ml
RB
0.5
.
—
0 .5
mi n
hr
5
IO
15
5
to
15
AAymin
0.0052
0.0022
----
0.0040
0.0028
-35-
P r o te c tio n a g a i n s t th e p h o to o x id a tio n b y h ip p u r y l - L p h e n y l a l a n i n e . A 0.1 M h ip p u r y l - L - p h e n y l a l a n i n e s o lu tio n in 0.1 M
T r i s , 1 ,0 M N a C l , pH 7 . 5 w a s p re p a re d b y d i s s o l v i n g 32 7 mg of
h ip p u r y l - L - p h e n y l a l a n i n e in e n o u g h o f th e b u ffe r to g iv e a t o t a l volum e
_O
o f 10 m l. O r th o - p h e n a n th r o lin e 2 x 10
M a n d m e th y le n e b lu e (0.199
m g/m l) w e re d i s s o l v e d in 0 . 1 M Tris b u ff e r, pH 7 . 5 , c o n ta i n in g 1.0 M
N a C l , a s p r e v io u s ly d e s c r i b e d .
As in p r e v io u s e x p e r im e n t s , th e
,
r e v e r s a l of th e in h ib iti o n d u e to th e m e ta l c h e l a t i n g a g e n t w a s
a c c o m p l i s h e d b y th e a d d itio n o f a s m a ll a liq u o t o f 0.1 M Z n C lg .
The
fin a l e n z y m e c o n c e n tr a tio n in a l l e x p e r im e n ta l s o lu tio n s w a s 0 . 0 8
m g /m l.
The a s s a y w a s a c c o m p lis h e d in th e fo llo w in g m anner;
a
b u ffe re d s o lu tio n w a s p r e p a r e d a t th e s a m e c o n c e n tr a tio n a s t h a t
n o rm a lly u s e d in p re p a rin g th e s u b s t r a t e s o l u t i o n , b u t th e s u b s t r a t e
w a s p u r p o s e ly o m itte d .
Th u s , w h e n a 50 m ic r o lite r a l i q u o t o f th e
e x p e r im e n ta l s o lu tio n c o n ta i n in g a la rg e e x c e s s o f s u b s t r a t e w a s
in tr o d u c e d in to t h i s b u f f e r , th e f in a l s u b s t r a t e c o n c e n tr a tio n o b ta in e d
“3
by d ilu tio n w a s 10
M w h ic h is th e n o rm al c o n c e n tr a tio n u s e d in a ll
assays.
P h o to o x id a tio n w a s a c c o m p lis h e d by M eth o d A.
T a b le XXII
s u m m a riz e s th e e x p e r im e n ta l c o m p o s itio n a n d th e r e s u l t s o f e a c h
d e term in a tio n .
S u b s tr a te p r o te c tio n in th e R ose B engal s y s t e m .
A h ip p u r y l- L -
p h e n y la l a n in e s o lu tio n in 0 . 0 3 M Tris b u f f e r , pH 7 . 4 , c o n ta i n in g 0 .5
M N a C l b u ff e r, w a s p r e p a r e d a s fo llo w s ;
3 4 , 6 mg of h ip p u r y l - L - p h e n y l ­
a l a n i n e w a s p l a c e d in a 10 ml v o lu m e tric f l a s k a n d d ilu te d to v o lu m e
—3
w ith a 10
M h ip p u r y l - L - p h e n y l a l a n i n e s o lu tio n in 0 . 0 2 5 M Tris b u ff e r,
pH 7 . 5 , c o n ta i n in g 0 . 5 M N a C l . R ose B engal w a s a d d e d to th is
s o lu tio n s o t h a t th e fin a l c o n c e n tr a tio n of d y e w a s 0 . 0 4 m g /m l.
Ten
m ic r o lite r s of a c a r b o x y p e p t i d a s e -Ay s o lu tio n th e n w a s d i l u t e d to 1.0
ml w ith t h i s b u ffe re d s y s te m a n d p h o to o x id iz e d for v a r io u s p e r i o d s of
t i m e . The p e p t i d a s e a c t i v i t y w a s d e te r m in e d by re m o v in g a t e n m ic ro ­
l i t e r a l i q u o t a n d a s s a y i n g in th e n o rm al m a n n e r .
The p h o to o x id a tio n w a s
d o n e a c c o r d in g to M e th o d B. The r e s u l t s a re s u m m a riz e d in T ab le XXIII.
-3 6 TABLE XXII
; y -i
unzymo
op
ml
sub.
20
0.36
—
O
<M
0 .3 6
—
20
0.36
■-----
20
0.36 I
C
D
E
T]
;
I
.
I
I
ml
min
hiry
—
— ’■■■
IO
1.64
25.3
...—
—
1 .6 4
2.5
1.0
3 0
—
0 .6 4
4.1
-----
1.0
30
10
0.64
$ 0.36
0.64
1 .0
—
IO
22.S
0.3 S
0.64
1.0
iO
16.9
ml
buffer
spec. cet.
pcpiidcse
__
20
O
\
zrr_T.%'
L
U
1
I * '
ml
____
P r o te c tio n a g a i n s t th e P h o to o x id a tio n
by H ip p u r y l- L - P h e n y la la n in e
30
7.7
The i n i t i a l c o n c e n t r a t i o n s o f r e a g e n t s d i s s o l v e d in 0.1 M Tris b u ff e r, pH
7 . 0 , c o n ta i n in g 1 .0 M N a C l w ere: OP 2 x 10
M; s u b s t r a t e , 0.01 M;
0.199 m g /m l; Z n C l2 , 0.1 M .
I
-37TABLE XXIII
S u b s tr a te P r o te c tio n R ose B engal S y ste m
y sl enzyme
_
sub
buffer I
mi n
hxr
trio
buffer
cpoe. eet .
peptidase
1.0
68a
1.0
80 .5
1.0
I
23.6
I
-3 8 -
P h o to o x id a tio n in th e p r e s e n c e o f g - p h e n y lp r o p io n a te . In to a s m a ll
p l a s t i c cup w a s p l a c e d 950 ^l o f a 0 .1 M Tris b u f f e r , pH 7 . 0 , c o n ta in in g
1 .0 M N a C l a n d 0 . 0 3 m g /m l o f Rose B e n g a l.
_o
Tris b u f f e r , pH 7 . 0 , c o n ta i n in g 2 x 10
Fifty m ic r o lit e r s o f a 0.1 M
M g -p h e n y lp ro p io n a te a n d 1 .0 M
N a C l w a s m ix e d w ith th e Rose .Bengal b u ff e r.
A c o n tro l b u ffe r s o lu tio n
c o n ta i n in g no jB -p h e n y lp ro p io n a te w a s p r e p a r e d a s a b o v e .
To e a c h s o lu tio n
10 [il of a c a r b o x y p e p tid a s e - A y s o lu tio n ( 8. 1 m g/m l) w a s a d d e d in th e d a r k .
E ach s o lu tio n w a s a s s a y e d for e s t e r a s e a c t i v i t y - i n th e u s u a l m a n n e r a n d
th e n e x p o s e d for v a r io u s p e rio d s o f tim e to a 500 w a tt lig h t s o u r c e .
e n z y m e w a s p h o to o x id iz e d a c c o r d in g to M e th o d B.
The
T a b le s XXIV and XXV
s u m m a riz e t h e - r e s u l t s o f tw o s e p a r a t e d e t e r m i n a t i o n s .
G ly c y lty r o s in e p r o t e c t i o n .
G l y c y l - L . - t y r o s i n e , 6 .3 m g, w a s
d i s s o l v e d in 0 .1 M Tris b u f f e r , -pH 7 . 5 , c o n ta i n in g 1 .0 M N a C l . The
f in a l c o n c e n tr a tio n o f g l y c y l - L - t y r o s i n e w a s 0. 001 M ;
N in e hu n d red
e ig h ty m ic r o lite r s of t h i s b u ffe re d s o lu tio n w a s th e n a d d e d to a s m a ll
p l a s t i c cup a n d 10 yl of a Rose B engal w a t e r s o lu tio n (300 m g/100 ml)
w as added.
Ten m ic r o lit e r s o f a c a r b o x y p e p tid a s e - A y s o l u t i o n (8. 1 m g/m l)
w a s a d d e d , a n d th e r e s u l t i n g s o lu tio n k e p t in th e d a r k .
The p e p t i d a s e
-
a c t i v i t y w a s d e te r m in e d a n d t h e n th e s o lu tio n w a s e x p o s e d to lig h t for
v a r io u s p e r io d s o f t i m e .
B-.
The lig h t s o u r c e w a s t h a t d e s c r i b e d in M eth o d
The p e p t i d a s e a c t i v i t y w a s m e a s u r e d a s d e s c r i b e d in " M e th o d s " ,
u s in g h ip p u r y l - L - p h e n y l a l a n i n e a s s u b s t r a t e .
T ab le XXVI s u m m a riz e s
th e r e s u l t s o f t h e s e d e te r m in a tio n s .
A g g re g a tio n s u b s e g u e n t to p h o to o x id a tio n of c a r b o x y p e p t i d a s e - A y .
A s p e c i a l co lu m n w a s p re p a re d in a n a tte m p t to rem ove th e s e n s i t i z i n g
d y e a n d a l s o to d e te r m in e I f a n y c o n fo rm a tio n a l c h a n g e o c c u r re d s u b s e ­
q u e n t to p h o to o x id a tio n of c a r b o x y p e p tid a s e - A y .
p re p a re d a s fo llo w s :
The co lu m n w a s
a 5 mm x 40 cm co lu m n w a s p a c k e d w ith 2 7 cm of
Seph'adex G - 7 5 , w h ic h h a d b e e n s lu r r ie d in 0 . 1 M Tris b u ff e r, pH 7 . 5 ,
c o n ta i n in g 1 .0 M N a C l . T his r e s i n th e n w a s c a p p e d w ith a . 3. 5 cm
s e c t i o n o f TEAE c e l l u l o s e r e s i n a l s o s u s p e n d e d in 0.1 M Tris b u ffe r.
-39TABLE XXIV
P h o to o x id a tio n in th e p r e s e n c e o f p -P h e n y lp ro p io n a te
?----------------------------------------r---------------------------------------min
hr
.
projected
I
I
5
10
-
20
I_ _ _ _ ! ! _ _ _ _
control
941
,S
15 7 0
1000
jj
755
a- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S
650
930
^
302
823'
I
52 6
-
994
|
|
-40-
TABLE XXV
P h o to o x id a tio n in th e p r e s e n c e
o f g - P h e n y lp r o p io n a te
min hv*
I
s p e c . a c t . e st cra so
0
I ISO
0
1515
0
I47 0
I
1340
1300
I
I
50
I
568
-4 1 -
TABLE XXVI
G ly c y lty r o s in e P r o te c tio n
min h v
s p e c . GCf. poptidcco
I
8 4 .8 ,8 4 .4
--------- ------ -----------------------------20
74.0
30
6 7 .4
130
54.6
I
—42 —
The e n tir e co lu m n w a s j a c k e t e d w ith c i r c u l a t i n g w a te r a t 0 ° c .
The e lu tio n
o f p r o te in w a s fo llo w e d b y e m p lo y in g th e flo w c e l l o f th e G ilfo rd s p e c t r o ­
p h o to m e te r w h ic h w a s s e t to m e a s u re t h e a b s o r b a n c e a t 2 78 m|_u The
e lu tio n p a t t e r n s o f o x id iz e d a n d u n o x id iz e d c a r b o x y p e p t i d a s e -Ay a re sh o w n
in F ig u re s I , 2 , a n d 3 r e s p e c t i v e l y .
C h ro m a to g ra p h y ''o f o x id iz e d a n d u n o x id iz e d c a r b o x y p e p tld a s e on
TEAE c e l l u l o s e . A co lu m n (31 x O05 cm) w a s p a c k e d w ith a s lu rry o f TEAE
c e l l u l o s e in 0 ..I M Tris b u ff e r, pH 7 . 5 , .c o n ta in in g 1 .0 M N a C L
Fifty
m ic r o lit e r s of a c a r b o x y p e p t i d a s e -Ay s o lu tio n ( 9. 21 m g /m l) w a s d ilu te d
w ith 200 |j.l o f e i t h e r 0..1 M Tris b u ff e r, pH 7 . 5 , c o n ta i n in g I. 0 M N a C l ,
or 0.1 M Tris b u ff e r, pH 7 . 5 , c o n ta i n in g L O M N a C l a n d 0 . 0 4 9 m g /m l of
R ose B en g a l. .The c u r v e s o f F ig u re s 4 , 5 , 6, a n d 7 s h o w th e e lu tio n
p a t t e r n s a n d a c t i v i t y p r o f ile s of o x id iz e d a n d u n o x id iz e d e n z y m e .
C h ro m a to g ra p h y o f p h o to o x id iz e d c a r b o x y p e p t l d a s e -A y on S e p h a d e x
G -7 5 c o lu m n . A S e p h a d e x G - 75 co lu m n (I cm x 25 cm) w a s p re p a re d w ith
0.1 M T r i s , .1.0 M N a C l , pH 7 . 5 b u f f e r .
The colum n w a s j a c k e t e d so t h a t
i t c o u ld be c o o le d to n e a r 0 ° C . w h e n u s e d in a norm al te m p e ra tu re r o o m .
The flo w r a t e w a s c o n tr o lle d b y pum ping th e b u ffe r a n d w a s d e te rm in e d
b e fo re a n d a f t e r e a c h ru n on th e c o lu m n .
fo llo w in g m anner:
S a m p le s w e re p r e p a r e d in th e
50 |j.l o f a c a r b o x y p e p t i d a s e ^Ay s o lu tio n ( 9. 21 m g/m l)
w e re d ilu te d w ith 200 ja l of 0.1 M Tris b u ffe r c o n ta in in g 0 . 0 4 9 m g /m l Rose
B e n g a l, pH 7 . 5 .
The s a m p le s w e re t h e n a s s a y e d for p e p t i d a s e a c t i v i t y ,
p h o to o x id iz e d , r e a s s a y e d , a n d p l a c e d on th e c o lu m n .
The r e s u l t s of
t h e s e d e te r m in a tio n s a re sh o w n in th e e l u tio n p a tte r n s o f F ig u re s 8 , 9 ,
a n d 10.
b e e n sh o w n to a c t i v a t e c a r b o x y p e p t i d a s e - A y , r e s u l t i n g in a tw o fo ld
2+
c o n tain i n c r e a s e in p e p t i d a s e a c t i v i t y , a s c o m p a re d to th e n a tiv e Zn
in g e n z y m e .
The i n a c t i v a t i o n o f th e e n zy m e w ith th e m e ta l c h e la to r
1 ,1 0 - p h e n a n th r o lin e h a s b e e n d e m o n s t r a t e d . This i n a c t i v a t i o n is a lm o s t
c o m p le te ly r e v e r s i b l e u p o n th e a d d itio n o f m e ta l i o n .
G e n e r a lly
th e
-43 —
0 .0 0 0
8.0
ml
c l uont
F ig u re I. S e p h a d e x G -7 5 c h ro m a to g ra p h y o f c a r b o x y p e p tid a s e - A Y
p h o to o x id ize d tw e n ty m in u te s.
-4 4 -
0.E75
0.025
m!
oluont
F ig u re 2 . S e p h a d e x G -7 5 c h ro m a to g ra p h y o f c a rb o x y p e p tid a s e - A y
p h o to o x id ize d s ix ty m in u te s .
-45 —
ml
eluonv
F ig u re 3 . S e p h a d e x G - 7 5 c h ro m a to g r a p h y o f c a r b o x y p e p ti d a s e - A y
u n o x id iz e d .
—
4 6—
optical
d e n c i t y 2 7 8 m/j
u n o Ht d i z e d
0.00
15.0
r r.I
c l u o n t
F ig u re 4 . C h ro m a to g ra p h y o f u n o x id iz e d c a r b o x y p e p t i d a s e -A y on
TEAE c e l l u l o s e .
-4 7 -
optical
density 278mu
O K i d i z C d
ml
eluent
Figure 5. C h ro m a to g ra p h y o f o x id iz e d c a r b o x y p e p t i d a s e o n TEAE
c e l l u l o s e , p h o to o x id iz e d tw e n ty m i n u t e s .
—4 8 —
optical
doncity
2 7 8 mju
oxidized
12D
mi
i
eluent
Figure 6. C h ro m a to g ra p h y o f o x id iz e d c a r b o x y p e p tid a s e o n TEAE
c e l l u l o s e . P h o to o x id iz e d t h i r t y m in u te s . (o -o ) i n d i c a t e s a c t i v i t y
p ro f ile o f p e a k s .
—49 —
0.00
ml
Fig u re 7.
c e llu lo se
Q Eu o nt
C h ro m a to g ra p h y o f u n o x id iz e d c a r b o x y p e p t i d a s e - A y o n TEAE
(o -o ) i n d i c a t e s a c t i v i t y p ro f ile o f p e a k s .
-50-
0.0E "
50
ml OlUGFlt
F ig u re 8. C h ro m a to g ra p h y o f n a tiv e c a r b o x y p e p t i d a s e - A y on S e p h a d e x
G -7 5 c o lu m n .
-51-
*2.7
3i
ml
Qluont
Fig u re 9 . C h ro m a to g ra p h y o f c a r b o x y p e p t i d a s e - A y o n S e p h a d e x
G -7 5 c o lu m n . P h o to o x id iz e d for s e v e n t y m in u te s b e fo re
c h r o m a to g r a p h y .
-5 2 -
26.7
m l
o i U Q R t
F ig u re 10. C h ro m a to g ra p h y o f c a r b o x y p e p t i d a s e - A y on S e p h a d e x G -7 5
c o lu m n . P h o to o x id iz e d for o n e h u n d re d tw e n ty m in u te s b e fo re b e in g
p l a c e d on c o lu m n .
-53-
r e a c t i v a t i o n h a s b e e n c a r rie d o u t w ith th e a d d itio n of s u f f i c i e n t Zn2+ in
0.1 M T r i s , I „.0 M N a C l , pH 7 . 0 , to t h e i n a c t i v e s y s t e m .
In th is s e r i e s
o f e x p e r i m e n t s , C o 2+ in 0.1 M T r i s , 1 .0 M N a C l , pH 7 . 0 w a s u s e d to
r e a c t i v a t e th e s y s te m s u b s e q u e n t to p h o to o x id a tio n .
As in p re v io u s
e x p e r i m e n t s , m e th y le n e b lu e a n d I ,.1 0 -p h e n a n th ro lin e w e re u s e d in fin a l
c o n c e n t r a t i o n s of 0 . 0 0 2 m g /m l a n d 5 x IO- 4 M r e s p e c t i v e l y .
n iq u e u s e d in t h e p h o to o x id a tio n w a s t h a t o f M eth o d A.
The t e c h ­
A ll-r e a c tio n s
w e re c a r r ie d o u t a t 0 ° C . P e p t i d a s e a c t i v i t y w a s d e te r m in e d a s d e s c r i b e d
in " M e t h o d s " , u s i n g h ip p u r y l - L - p h e n y l a l a n i n e a s s u b s t r a t e . T ab le XKVII
s u m m a riz e s th e e x p e r im e n ta l c o m p o s itio n a n d r e s u l t s o f e a c h d e t e r m i n a t i o n .
P r e p a r a tio n o f c o b a l t - c a r b o x y p e p t i d a s e -Ay
b y
d ia ly sis. A
s o lu tio n of c a r b o x y p e p tid a s e - A y ( 3 . 5 0 m g /m l) d i s s o l v e d in 0 . 0 5 M T r i s ,
0 . 0 5 M ,sodium a c e t a t e , 1 .0 M N a C l , pH 7 . 9 5 a t S0 C . w a s p la c e d in a
d i a l y s i s b a g w h ic h h a d b e e n p r e v i o u s l y t r e a t e d w ith h o t w a t e r , s o a k e d
in d e i o n i z e d w a t e r a n d in 0 . 0 5 M T r i s , 0 . 0 5 M so d iu m a c e t a t e , 1 .0 M
—2
N a C l , pH 8 . 0 b u ffe r c o n ta i n in g 10
M C o C l9 . D i a l y s i s w a s c o n tin u e d
2+ ^
in t h i s b u ffe r for 48 h o u r s , a f t e r w h ic h Co ■ free b u ffe r w a s s u b s t i t u t e d .
F o llo w in g s e v e n h o u rs in t h e m e ta l fre e b u f f e r , a s e c o n d c h a n g e o f m e ta l
fre e b u ffe r w a s in tr o d u c e d a n d d i a l y s i s c o n tin u e d a n a d d i t i o n a l 24 h o u r s .
At t h i s p o in t th e p r o te in c o n c e n tr a tio n w a s d e te rm in e d b y m e a s u rin g th e
o p t i c a l d e n s i t y a t 2 78 mp..
A ssu m in g th e s a m e a b s o r p t i v i t y a s th e n a tiv e
z i n c e n z y m e , th e c o n c e n tr a tio n w a s 2 . 7 6 m g /m l.
A 5 pi a l i q u o t o f th is
p r e p a r a t io n w a s t h e n d ilu te d to 1 .0 ml a n d a 50 pi p o rtio n a s s a y e d for
p e p t i d a s e a c t i v i t y in th e u s u a l m a n n e r.
The s p e c i f i c a c t i v i t y w a s d e ­
te rm in e d to be 9 9 . 4 u n i t s / m g a s c o m p a re d to th e 6 9 . 5 u n i t s / m g of th e
n a tiv e e n z y m e , or a 1.4 3 fo ld i n c r e a s e in p e p t i d a s e a c t i v i t y .
P h o to o x id a tio n o f c o b a l t - c a r b o x y p e p t i d a s e - A y a n d n a tiv e
c a r b o x y p e p t i d a s e - A y in s a m e b u ffe r s y s t e m .
C o b q lt-carb o x y p ep tid ase-A y
w a s p r e p a r e d b y d i a l y s i s a g a i n s t 0.1 M T r i s , 1.0 M N a C l , b u ffe r c o n t a i n O
ing-10
M C oC l^ for 48 h o u rs . The a c t i v i t y o f th e c o b a l t - c a r b o x y p e p t i d a s e - A y w a s d e te r m in e d a n d found to be 1 .5 tim e s t h a t of th e n a tiv e
-5 4 TABLE XXVII
Co
I
A c tiv a tio n o f P h o to o x id iz e d C a r b o x y p e p ti d a s e - A y
ml
buffer
enzyme
A
B
I
i
\\
!!
ml
OP
ml
MB
j min
Snrr
I
|
5
5
I 0I
5
0
5
— ■
|
I
1.0
I
I
0.75
0.25
0.75
0.2 5
0.75
0 .2 5
I
E
I
6
0.25
0.25
r
I
6
0.25
0.25
—
0.5
spec. cot.
! peptidase
——
61.3
—
—
—
0.5
I
________ I
yul
Co
4.0
5
-----
479
—
IO
105.5
30
5
30
—
—
IO
27.9
53.8
The r e a g e n t s w e re d i s s o l v e d in 0.1 M Tris b u ff e r, pH 7 . 0 , c o n ta i n in g 1 .0 M
H a Q a t i n i t i a l c o n c e n t r a t i o n s of: O P, 2 x IO-3 M; MB, 0 . 0 0 4 m g /m l; ZnCL1,
0 .1 M; C o C l2 0.1 M .
^
-5 5 -
enzym e.
This c o b a l t e n z y m e p r e p a r a t io n w a s n o t fre e d o f a d v e n ti tio u s
m e ta l i o n s .
fo llo w s :
P h o to o x id a tio n o f th e c o b a l t - c a r b o x y p e p t i d a s e - A y w a s a s
10 ij.I o f th e c o b a l t a n d c a r b o x y p e p tid a s e - A y ( 4. 03' m g/m l) w a s
d i l u t e d w ith 1.0 ml o f 0 .1 M T r i s , 1.0 M N a C l , pH 7 . 5 b u ffe r a t 4 ° C .
F ifty m ic r o lite r s o f a m e th y le n e b lu e w a t e r s o lu tio n ( a p p ro x im a te ly 3 . 0
m g /m l) w a s a d d e d to th e en zy m e s o l u t i o n .
The m ix tu re w a s d i v i d e d , a n d
one h a l f s to r e d in t h e d a r k , w h ile th e s e c o n d h a lf w a s e x p o s e d to th ir ty
m in u te s o f l i g h t .
The p h o to o x id a tio n w a s d o n e u s i n g M e th o d A.
P ep ti­
d a s e a c t i v i t y w a s d e te r m in e d a s d e s c r i b e d in " M e th o d s " , u s i n g
h ip p u r y l - L - p h e n y l a l a n i n e a s s u b s t r a t e .
The r e s u l t s of t h i s d e te r m in a tio n
a re s u m m a riz e d in T ab le XXVIIl,' p a rt A.
P a rt B of Table XXVIII s u m m a riz e s
th e r e s u l t s o f a n i d e n t i c a l tr e a tm e n t o f a n a tiv e c a r b o x y p e p t i d a s e s o l u t i o n .
P r e p a r a tio n a n d p h o to o x id a tio n o f n i c k e l - c a r b o x y p e p t i d a s e - A , A
1.5 ml a l i q u o t o f a c a r b o x y p e p tid a s e - A y s o lu tio n ( 2 . 6 7 m g/m l) in 0 . 1 M
T r i s , 1 .0 M N a C l , pH 7 . 5 b u ffe r w a s p l a c e d in a p r e t r e a t e d d i a l y s i s
bag.
The d i a l y s i s b a g th e n w a s im m e rs e d in o n e h u n d re d ml o f 0.1 M
T r i s , 1.0 M N a C l , 10 ^ M N iC lg , pH 7 . 5 3 b u ffe r a n d d i a l y s i s c o n tin u e d
24 h o u r s . P h o to o x id a tio n s t u d i e s on t h i s s o lu tio n w e re a s fo llo w s :
50
pal o f th e n i c k e l - c a r b o x y p e p t i d a s e - A y s o lu tio n w e re d i lu te d w ith I ml of
0.1 M Tri s , 1 .0 M N a C l , b u ffe r a n d 50 pl o f a m e th y le n e b l u e - w a t e r
s o lu tio n (a p p ro x im a te ly 4 . 0 m g/m l) w a s a d d e d .
The s o lu tio n w a s
d i v i d e d , a n d o n e h a l f e x p o s e d to 30 m in u te s o f l i g h t .
w a s s to r e d in th e d a rk for a s im ila r le n g th of tim e .
w a s b y M e th o d A.
The s e c o n d h a lf
The p h o to o x id a tio n
P e p t i d a s e a c t i v i t y w a s d e te rm in e d a s d e s c r i b e d in
" M e t h o d s " , u s i n g h ip p u r y l - L - p h e n y l a l a n i n e a s s u b s t r a t e . The r e s u l t s
o f t h i s p h o to o x id a tio n a re s u m m a riz e d in T a b le XXIX p a r ts A a n d B.
P a rts C a n d D o f T ab le XXIX s u m m a riz e th e d a ta c o l l e c t e d w h e n n i c k e l c a r b o x y p e p tid a s e - A y i s p h o to o x id iz e d u n d e r i d e n t i c a l c o n d i t i o n s , bu t
w ith th e s e n s i t i z e r R ose B e n g a l.
C o p p e r C a r b o x y p e p ti d a s e - A y.
C o p p e r - c a r b o x y p e p tid a s e - A y
w a s p r e p a r e d b y d i a l y s i s o f n a tiv e z i n c c a r b o x y p e p tid a s e - A y ( 2 . 6 7 m g/m l)
-56-
TABLE XXVIII
P h o to o x id a tio n o f C o b a lt-C a r b o x y p e p tid a s e - A y an d
N a tiv e C a r b o x y p e p tid a s e - A y in sa m e b u ffe r s y s te m
part
I
hal f
|
1
I
A
A
min
hiT
A A y / min 2 5 4 m/u.
0.0214
I
0.0334
j-I
J
30
0.0202
------
0.0256
B
2
;
-5 7 TABLE XXDC
P re p a ra tio n a n d P h o to o x id a tio n o f N ic k e l- C a r b o x y p e p tid a s e
hclf
port
min
fnr'
|
mfn 2 5 4 m,u.
0.01 G4
2
B
C
..J I
l
I
>
___ .
D
-----I
I
0.0 2 1 0
I
30
)
0.0033
I_______________________?
2
j
--------
I
0.0200
-5 8 -
in 0.1 M T r is , 1.0 M N a C l, pH 7 .5 b u ffe r a g a in s t 100 ml o f th e sam e
_o
b u ff e r, c o n ta in in g 10
M C oC lg , fo r 24 h o u rs , an d w a s fo llo w e d by tw o
c h a n g e s o f m e ta l free b u ffe r.
F ifty m ic ro lite rs o f th e c o p p e r - c a r b o x y p e p tid a s e w e re rem o v ed
from th e d ia ly s is b a g a n d d ilu te d to 1 .0 ml w ith Tris. b u ffe r pH 7 . 5 .
s p e c if ic p e p tid a s e a c ti v ity o f th is s o lu tio n w a s 6 . 2 .
The
W hen th is s o lu tio n
__ O
w a s fu rth e r d ilu te d w ith o n e ml o f T ris b u ffe r, pH 7 . 5 , c o n ta in in g 2 x 10
M 1 , 1 0 -p h e n a n th ro lin e an d 20 p,l o f 10
-M ZnClg s o lu tio n a d d e d , th e
s p e c if ic a c ti v ity in c r e a s e d to 61.0 u n its /m g .
T his p ro c e d u re w a s th e n
u s e d in a s s a y in g th e c o p p e r -c a rb o x y p e p tid a s e -A y s u b s e q u e n t to p h o to ­
o x id a tio n or o th e r c h e m ic a l m o d ific a tio n s .
P h o to o x id a tio n o f c o p p e r ^ c a rb o x y p e p tid a s e -A y w a s d o n e a s
fo llo w s:
50 [il o f c o p p e r c a rb o x y p e p tid a s e -A y p re p a re d b y d ia ly s is w a s
d ilu te d to 1 .0 ml w ith 0-..1 M T r is , .1.0 M N a C l, pH 7 .5 b u ffe r. .F ifty
m ic ro lite rs o f a R ose B e n g a l/w a te r s o lu tio n , (a p p ro x im a te ly 3 m g/m l)
w a s a d d e d to th e d ilu te d en zy m e s o lu tio n a n d th e sa m p le d iv id e d .
O ne
h a lf w a s e x p o s e d to 30 m in u te s o f lig h t, a n d th e o th e r s to re d in th e d a r k .
M eth o d A w a s u s e d to p h o to o x id iz.e th e e n z y m e .
The a s s a y fo r p e p tid a s e
a c ti v ity w a s d o n e a s d e s c r ib e d a b o v e , u s in g h ip p u ry l-L -p h e n y la la n in e a s
s u b s tra te -.
m e n t.
The d a ta in T ab le XXX-A su m m a riz e th e -re su lts o f th is e x p e r i­
The d a ta in T a b le XXX-B su m m a riz e th e r e s u lts o f a r e p e a t of th e
a b o v e e x p e rim e n t.
. E ffe c t o f R ose B engal on th e e x c h a n g e o f Cu
c a r b o x y p e p tid a s e - A y.
2+
a n d Zn
2+
io n s in
F ifty m ic ro lite rs o f c o p p e r -c a rb o x y p e p tid a s e -A y
w h ic h h a d b.een p re p a re d by d ia ly s is w a s d ilu te d to a c o n c e n tra tio n of
0 .1 3 4 m g /m l w ith 0 .1 M T ris , I. 0 M N a C l, pH 7 .5 b u ffe r.
A w a te r
s o lu tio n o f R ose B engal (3 m g/m l) w a s p r e p a r e d . A liq u o ts o f th is R ose
B engal s o lu tio n w e re a d d e d to s e v e r a l o f th e en zy m e s o lu tio n s , and th e
a b ility o f Zn^+ io n s to r e a c tiv a te th e e n zy m e w a s d e te rm in e d . R e a c ti_g
v a tio n w a s a c c o m p lis h e d by a d d in g -1 .0 ml o f a 2 x 10
M 1, 1 0 -p h e n a n th ro ­
lin e s o lu tio n in 0 .1 M T r i s , .1.0 M N a C l, pH 7 .5 b u ffe r, fo llo w e d by
-5 9 -
TABLE XXX-A
C o p p e r C a rb o x y p e p tid a s e -A Y
ml
buffer
L enzyme
50
50
RA
—
50
OPiZnaddod
s p e c . act.
peptidaso
VOG
yoo
15.2
28.4
1.0
30
1.0
—
yos
61.0
1.0
—
no
6.2
-
-
TABLE XXX-B
ml
buffor
OP1Zn added
-
50
50
50
—
1.0
-
-
.
....................... -
30
—
.
.
I
I
__________________
yos
yos
O
RS
I
>*J
I
enzymo 2
b
I
min
.
-
- .
I
j
spoc. act. I
pcptidcso I
14.9
22.4
4.36
-6 0 -
2 0 |_il o f a 0 . 1 M ZnClg s o lu tio n in T ris b u ffe r.
P e p tid a s e a c tiv ity w as
d e te rm in e d a s d e s c r ib e d in " M e th o d s " , u s in g h ip p u ry l-L -p h e n y la la n in e
as s u b s tra te .
T ab le XXXI s u m m a riz e s th e r e s u lts o f t h e s e d e te r m in a tio n s ,
■S e p h a d e x G -7 5 c h ro m a to g ra p h y o f c a r b o x y p e p tid a s e - A y in th e
p r e s e n c e o f 10
M C u ^+ i o n s ,
__ O
1 .0 M N a C l, an d 10
O,
M Cu
T ris b u ffe r, 0 ..I M , pH 7 . 0 , c o n ta in in g
io n s , w as p re p a re d .
3 .9 mg o f R ose
B engal w a s a d d e d to th is b u ffe r, an d th e p h o to la b ility o f c a r b o x y p e p ti­
d a s e -A y in th is b u ffe r d e te rm in e d .
The lo s s o f p e p tid a s e a c tiv ity and
e s t e r a s e a c tiv ity a s a r e s u l t o f p h o to o x id a tio n in th is b u ffe r is show n
in T ab le XXXII.
T his d a ta w a s o b ta in e d w h en 10 pi o f a c a rb o x y p e p ti­
d a s e -A y s o lu tio n ( 9. 21 m g/m l) w a s d ilu te d w ith 500 pi o f th e C u^+ io n
c o n ta in in g b u ffe r.
P h o to o x id a tio n w a s d o n e a s d e s c r ib e d in " M e th o d s" .
The S e p h a d e x G -7 5 c h ro m a to g ra p h y w a s a c c o m p lis h e d in a I x 64 cm
colum n a t 0 ° c .
O ne h u n d re d m ic ro lite rs o f a c a r b o x y p e p tid a s e -A y
s o lu tio n (9.2.1 m g/m l) w a s d ilu te d w ith 500 pi o f th e R ose B engal
c o n ta in in g b u ffe r a b o v e an d p la c e d on th e c o lu m n .
The e n zy m e w as
th e n e lu te d from th e colum n w ith 0.1 M T ris b u ffe r, pH 7 . 0 , .c o n ta in in g
1 .0 M N a C l.
The e lu tio n p a tte rn o f o x id iz e d an d u n o x id iz e d c a rb o x y ­
p e p tid a s e -A y in th is b u ffe r s y s te m a re sh o w n in F ig u re s 11 a n d 12 .
P h o to o x id a tio n o f c o b a lt- c a r b o x y p e p tid a s e - A y.
C o b a lt-
c a r b o x y p e p tid a s e - A y p re p a re d by d ia ly s is in 0 . 0 5 M T ris b u f f e r , pH
8 . 0 , c o n ta in in g 0 . 0 5 M so d iu m a c e ta te a n d -1 .0 M N a C l, w a s p h o to o x id iz e d w ith m e th y le n e b lu e s e n s i t i z e r . .Five m ic ro lite rs o f th e c o b a lt
e n zy m e w e re d ilu te d to I . O1ml w ith 0 .5 ml o f 0 ..05 M T ris b u ffe r, pH
7 . 9 5 , . 0. 05 M so d iu m a c e ta te an d 1 .0 M N a C l, an d 0-. 5 ml o f 0.1 M
T r is , b u ff e r, pH 7 . 5 , c o n ta in in g 1 .0 M N a C l, an d 0 .1 9 8 m g /m l of
m e th y le n e b lu e .
The r e s u ltin g s o lu tio n w a s th e n d iv id e d , a n d one h a lf
k e p t in th e d a rk fo r-th irty m in u te s , w h ile th e o th e r h a lf w a s e x p o s e d to
th ir ty m in u te s o f l i g h t .
P h o to o x id a tio n w a s a c c o m p lis h e d b y M eth o d A.
-6 1 TABLE XXXI
sn
O
E ffe c t o f R ose B engal on th e E x c h a n g e o f C u ^+ an d
Zn^+ io n in C a r b o x y p e p tid a s e -A y
yul CuCPD
'----------------------------I
I
I
ml
buffer
spec,
peptidase
50
0
1.0
72.3
50
5
1.0
S 0.5
!0
10
50
20
1.0
492
SO 7
50
1.0
26.1
50
10 0
1.0
29.3
50
_
1.0
14.3*"*
SG
5 9 .3
A s s a y w a s p erfo rm ed w ith o u t th e a d d itio n o f b u ffe r c o n ta in in g OP an d 20 pi
o f Zn2+ b u ffe r.
-6 2 TABLE XXXII
P h o to o x id a tio n o f C a rb o x y p e p tid a s e -A y in th e
P r e s e n c e o f IO- ^ M Cu^+ io n s
min
hv
epee.
a c t . peptidase
53.9
epoe.
a c t . osfcraeo
278m#
density
optical
F ig u re 11. S e p h a d e x G -7 5 c h ro m a to g ra p h y o f c a rb o x y p e p tid a s e - A y in
th e p r e s e n c e o f IO "3 M C u2+ i o n s , p h o to o x id iz e d fo r s ix h o u r s .
—
64 —
0.00
ml
Qi uont
F ig u re 12. S e p h a d e x G -7 5 c h ro m a to g ra p h y o f c a r b o x y p e p tid a s e - A y in th e
p r e s e n c e o f 10- 3 m C u 2+ i o n s .
i
-6 5 -
The p e p tid a s e a c ti v ity w a s d e te rm in e d a s d e s c r ib e d in " M e th o d s " , u s in g
h ip p u ry l-L -p h e h y la la n in e a s s u b s t r a t e .
The r e s u lts o f t h e s e d e te rm i­
n a tio n s a re g iv e n in T ab le XXXIII.
P h o to o x id a tio n o f c a r b o x y p e p tid a s e - A y in 0 . 0 5 M so d iu m a c e t a t e ,
0 . 0 5 M T ris , 1.0 M N a C l b u f f e r .
B e c a u se o f th e r a th e r la rg e d e c r e a s e
in a c tiv ity o f th e c o b a lt- c a r b o x y p e p tid a s e w h en p h o to o x id iz e d in 0 . 0 5 M
so d iu m a c e t a t e , 0 . 0 5 M T r i s , 1 .0 M N a C l, an d 0.1 M T r i s , 1 .0 M N a C l,
0.199 m g /m l m e th y le n e b l u e , pH 7 .5 b u ffe r m ix tu re , i t w a s f e lt th a t th e
d e c r e a s e in th e a c ti v ity o f th e n a tiv e c a r b o x y p e p tid a s e in th is b u ffe r
s y s te m s h o u ld b e d e te rm in e d for c o m p a ris o n .
T h u s , 5 jjl o f a c a rb o x y ­
p e p tid a s e -A y s o l u t i o n , 4 . 0 3 m g /m l, w as d ilu te d w ith 0 .5 ml o f e a c h o f
th e a b o v e d e s c r ib e d b u f f e r s , an d th e sa m p le d iv id e d .
O ne h a lf w a s s to re d
in th e d a rk a t 0 ° C . , w h ile th e o th e r h a lf w a s e x p o s e d to 30 m in u te s of
lig h t.
The lig h t s o u rc e an d a s s a y c o n d itio n s .w e re a s p re v io u s ly d e ­
s c r ib e d in M eth o d A.
The r e s u lts a re su m m a riz ed in T ab le XXXIV-A.
In a s im ila r fa sh io n ., 10 |j.l o f c a rb o x y p e p tid a s e w a s d ilu te d w ith
0 .5 ml o f th e -m e th y le n e b lu e b u ffe r, a s d e s c r ib e d a b o v e , a n d 0 .5 ml o f
0 .1 M T r i s , I .) M N a C l, pH 7 .5 a t 0 ° C . b u f f e r . T his s o lu tio n lik e w is e
w a s d iv id e d , a n d one h a lf o x id iz e d 30 m in u te s .
The r e s u lts o f th is
d e te rm in a tio n a re sh o w n in T ab le XXXIV-B.
P h o to o x id a tio n o f c a rb o x y p e p tid a s e in a c e ta te io n b u f f e r . Ten
m ic r o lite r s o f a c a rb o x y p e p tid a s e -A y s o lu tio n ( 4 . 0 3 m g /m l) w a s d ilu te d
to 1 .0 ml by th e a d d itio n o f 0 .5 ml o f 0 . 0 5 M Tris b u f f e r , pH 7 . 9 5 , c o n ­
ta in in g 1 .0 M N a C l a n d 0 . 0 5 M so d iu m a c e t a t e , an d 0 .5 ml o f 0.1 M
T ris b u f f e r , pH 7 .0 a t 0 ° C . , c o n ta in in g 1 .0 M N aC l an d 0 .2 m g /m l
m e th y le n e b l u e .
The m ix tu re th e n w a s d iv id e d , an d o n e h a lf e x p o s e d to
30 m in u te s o f lig h t, w h ile th e s e c o n d h a lf w a s s to re d in th e d a r k .
P h o to o x id a tio n w a s done a c c o rd in g to M eth o d A.
P e p tid a s e a c tiv ity w as
d e te rm in e d a s d e s c r ib e d in " M e th o d s " , u s in g h ip p u ry l-L -p h e n y la la n in e
a s s u b s tr a te .
XXXV.
The r e s u lts o f th is e x p e rim e n t are. su m m a riz ed in T able
“66TABLE XXXIII
P h o to o x id a tio n o f C o b a lt- C a r b o x y p e p tid a s e - A y
ho|?
ml n
hxr
spec.
a c t . pcptfdcco
C4.C
I
30
2
I
41.2
-6 7 -
TABLE XXXIV
P h o to o x id a tio n o f C a r b o x y p e p tid a s e - A y in 0 . 0 5 M so d iu m
a c e t a t e , 0 . 0 5 M T r i s , 1 .0 M N aC l b u ffe r
h o If
I
min hv
_____________________
30
0 .0 1 2 6
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0 .0 2 42
_ _ _ _ _ I_ _ _ _ _ I
2
h a lf
I
2 5 4 m/4
min
30
2
hir
A / y l n i n 2 5 4 nyx
________ _ -0.0170
0.0210
-
- 6 8 -
TABLE XXXV
P h o to o x id a tio n o f C a r b o x y p e p tid a s e
in A c e ta te Io n Buffer
I
h alf
mi n
hiM
A /y'm in 2 5 4
I
30
0.0118
2
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0.0233
—69 —
P h o to o x ld a tio n o f c a rb o x y p e p ti d a s e -A y w ith R ose B engal and
d e te rm in a tio n o f s e d im e n ta tio n v e lo c ity .
A 2 00 yl p o rtio n o f a c a rb o x y -
p e p tid a s e -A y s o lu tio n (2 8 .1 4 m g/m l) d is s o lv e d in 3 M N a C l w a s d ilu te d
w ith 0 .8 ml o f 0 . 1 M Tri s b u ffe r, pH 7 .5 a t 4 ° C . , c o n ta in in g 0 . 7 7 M
N a C l. A s m a ll q u a n tity o f R ose B engal (u n m e a su red ) w a s a d d e d to th e
s o lu tio n .
The p e p tid a s e a c ti v ity o f th is s o lu tio n w as th e n d e te rm in e d
in th e u s u a l m a n n e r by rem ov ng a 10 pi a liq u o t a n d d ilu tin g i t to 1 .0 ml
w ith 0.1 M T ris b u f f e r . A 50 pi a liq u o t.o f th is s o lu tio n w a s a s s a y e d fo r
p e p tid a s e a c ti v ity .
The re m a in d e r o f th e en zy m e s o lu tio n w a s p h o to -
o x id iz e d fo r 2 .1/2 h o u r s . The e n zy m e w a s p h o to o x id iz e d a c c o rd in g to
M eth o d A.
The in itia l s p e c if ic a c ti v ity o f th e en zy m e s o lu tio n w as ■
d e te rm in e d to be 5 3 . 4 u n its /m g , an d fo llo w in g 2 ,1/2 h o u rs o f p h o to ­
o x id a tio n w a s 2 2 . 6 u n its /m g
A 0 .6 ml p o rt on o f th e p h o to o x id iz e d
en zy m e s o lu tio n w a s th e n p la c e d in a s in g le s e c to r c e ll a n d th e
s e d im e n ta tio n v e lo c ity d e te rm in e d a t 6 0 , 0 0 0 rpm in th e B eckm an m odel
E u ltr a - c e n tr if u g e .
The m o st -s ig n ific a n t o b s e r v a tio n m ade from th is
s tu d y w a s th a t th e p h o to o x id iz e d e n zy m e d id n o t s e d im e n t a s a s in g le
s p e c i e s , b u t r a th e r a s a c o m p le te ly h e te ro g e n o u s s y s te m , in w h ic h none
o f th e c o m p o n e n ts w e re o f s u f f ic ie n t c o n c e n tra tio n to a p p e a r a s a s in g le
s e d im e n tin g b o u n d a ry .
P h o to o x id a tio n o f c a r b o x y p e p tid a s e - A y in v a rio u s b u ffe r s y s t e m s ,
The r a te o f in a c tiv a tio n a s a r e s u lt o f p h o to o x id a tio n in v a rio u s b u ffer
s y s te m s w a s s tu d ie d .
F ifty m ic ro lite rs o f a s to c k c a r b o x y p e p tid a s e -A y
s o lu tio n (8. 1 m g/m l) w a s d ilu te d w ith 6 ml o f th e fo llo w in g b u ffe r
s o lu tio n s :
0,1 M so d iu m c itr a te b u ffe r, pH 7 . 0 , c o n ta in in g 1 .0 M N aC l;
0.1 M T ris b u f f e r ,.pH 7 . 0 , c o n ta in in g 1 .0 M NaCl;. 0.1 M T ris b u ffe r,
pH 7 . 0 , c o n ta in in g 1 .0 M N a g S O ^ .
E ach b u ffe r s o lu tio n w a s d iv id e d
in to s ix p a r t s , a n d e a c h p a rt e x p o s e d to v a rio u s p e rio d s o f p h o to o x id a tio n .
The lig h t s o u rc e w a s th a t d e s c r ib e d in M eth o d B.
T a b le XXXVI .su m m a riz es
th e lo s s o f e s t e r a s e a c ti v ity .in th e v a rio u s -buffer s y s t e m s .
a c ti v ity w a s d e te rm in e d a s d e s c r ib e d in " M e th o d s " .
The e s t e r a s e
-7 0 TABLE XXXVI
P h o to o x icja tio n o f C a r b o x y p e p tid a s e -A y
in V a rio u s B uffer S y ste m s
spec.
act.
I
s po c .
564
act.
spec.
citrate
DISCUSSION AND CONCLUSIONS
The e x p e rim e n ta l d a ta d is c u s s e d in th is s e c tio n w e re s e le c te d
from th o s e e x p e rim e n ts w h ic h w ere p erfo rm ed a f te r the- e x p e rim e n ta l
m e th o d s h ad b e e n p e r f e c t e d .
M any, o f th e e x p e rim e n ts in th e p re v io u s
s e c tio n w e re o f a p ro b in g n a t u r e , an d s u b s e q u e n tly th e r e s u lts o b ta in e d
w e re in fe rio r to th o s e r e s u lts o b ta in e d l a t e r .
The re a d e r s h o u ld be a w a re
th a t tw o m e th o d s o f p h o to o x id a tio n w e re u s e d in o b ta in in g d a t a , and
s h o u ld n o t q u a n tita tiv e ly com pare d a ta o b ta in e d b y d iff e r e n t m e th o d s .
In a d d itio n , a tte n tio n s h o u ld be p a id to the. en zy m e c o n c e n tra tio n s of
th e v a rio u s e x p e r im e n ts , s in c e th e ra te o f o x id a tio n is c o n c e n tra tio n
d e p e n d e n t.
S in c e i t is n e c e s s a r y to p h o to o x id iz e b o th th e n a tiv e an d th e
a p o e n z y m e in o rd e r to d e te rm in e if h is tid in e w a s in v o lv e d in b in d in g
z in c , a c o n v e n ie n t m eth o d fo r th e p re p a ra tio n o f th e a p o e n z y m e h ad to
be fo u n d . V a lle e h a s p re v io u s ly d e s c r ib e d th e p re p a ra tio n o f
(3)
a p o c a rb o x y p e p tid a s e -A y on a S e p h a d e x G -2 5 c o l u m n /
H o w ev e r,
th is p re p a ra tio n is s ta b le o n ly o v e r s h o rt p e rio d s o f t i m e . In a d d itio n
to th e in s t a b i l i t y .o f th e a p o e n z y m e p re p a re d b y th e co lu m n m e th o d ,
o th e r in c o n v e n ie n c e s a re e n c o u n te re d w ith th is m e th o d .
The a p o en z y m e
is r e a c tiv a te d ra p id ly by th e m e ta l io n s w h ic h a re p r e s e n t in m inute
q u a n tity in th e p l a s t i c w a re a n d g l a s s w a re .
E xtrem e p r e c a u tio n s m u st
be e m p lo y e d to p re v e n t th is r e a c tiv a tio n , an d th e y w e re fo u n d to be to o
in c o n v e n ie n t to e m p lo y on a re g u la r b a s i s .
T h erefo re th e a p o en z y m e w as
p re p a re d by sim p ly in c u b a tin g th e n a tiv e en zy m e in th e p r e s e n c e of
1, 1 0 - p h e n a n th r o lin e .
The a p o e n z y m e p re p a re d in th is m a n n er c a n be
fu lly r e a c tiv a te d b y th e a d d itio n o f a s u ita b le q u a n tity o f z in c i o n s .
H av in g n ow a s y s te m in w h ic h one c a n r a p id ly p re p a re th e a p o e n z y m e ,
p h o to o x id a tio n w a s -u n d e rta k e n u s in g th e d y e m e th y le n e b lu e . The d a ta
in F ig u re 13 sh o w s th e e f f e c t o f th e p h o to o x id a tio n on th e p e p tid a s e
a c ti v ity .
The lo s s o f p e p tid a s e a c ti v ity fo llo w in g -a s im ila r p h o to o x i­
d a tio n o f th e n a tiv e e n zy m e is n o t .a p p r e c ia b le .
t h a t z in c b in d in g h a s a p ro te c tiv e e f f e c t.
T h e se r e s u lts s u g g e s t
The r e s u lts in F ig u re 14 re v e a l
a s trik in g d iff e r e n c e b e tw e e n th e e f f e c t o f p h o to o x id a tio n on th e gam m a
-7 2 -
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F igure 13. Bar A r e p r e s e n ts th e n a tiv e a c t i v i t y o f c a r b o x y p e p tid a s e - A y
a t a c o n c e n tra tio n o f 0 . 0 2 3 m g /m l. Bar B r e p r e s e n ts th e a c ti v ity
re m a in in g a f te r p h o to o x id a tio n o f th e n a tiv e en zy m e w ith m e th y le n e b l u e .
Bar C r e p r e s e n ts th e a c t i v i t y re m a in in g a f te r p h o to o x id a t o n o f th e e n ­
zym e w ith m e th y le n e b lu e in th e p r e s e n c e o f 5 x 10"^ M 1, 1 0 -p h e n a n th ro Iin e s u b s e q u e n t to r e a c tiv a tio n w ith IO- * M ZnClg b u ffe r. The m e th y le n e
b lu e c o n c e n tra tio n w a s 0 . 0 0 2 m g /m l in a ll c a s e s . P h o to o x id a tio n w as
c a r rie d o u t a c c o rd in g to M eth o d A.
-73-
O —p ep tid ase
X—e ste ra se
tim e
sec
F igure 14. P h o to o x id a tio n o f c a r b o x y p e p tid a s e - A y in 1 .0 M N a C l, 0.1
M T r i s , 0.10 m g /m l R ose B e n g a l, pH 7 . 5 , a t 3 ° C. P ro te in c o n c e n ­
tr a tio n 0 . 0 2 8 m g /m l. 500 w a tt lig h t s o u rc e fo c u s e d th ro u g h a n f 1 .6
le n s w a s u s e d . S a m p le s w e re p la c e d 4 .3 cm from th e l e n s . E s te ra s e
a c ti v ity (x -x ) a n d p e p tid a s e a c ti v ity (o -o ) w e re d e te rm in e d s p e c tr o p h o to m e tric a lly a s d e s c r ib e d in " M e th o d s " .
—74 —
form o f th e e n zy m e w h en c o m p ared to th e d e lta f o r m
Ph o t o o x i d a t l o n
o f 6 -c a rb o x y p e p tid a s e -A r e s u lts in a l o s s o f p e p tid a s e a c tiv ity , an d in a
tw o -f o ld in c r e a s e in th e e s t e r a s e a c t i v i t y .
The d a ta in F ig u re 14 in d ic a te s
th a t p h o to o x id a tio n w ith m e th y le n e b lu e o f a p o c a rb o x y p e p tid a s e -A y r e ­
d u c e s th e e s t e r a s e a c t i v i t y a t th e sa m e r a te a s th e p e p tid a s e a c tiv ity is
l o s t , w h ic h in d ic a te s a s ig n if ic a n t d iff e r e n c e b e tw e e n th e tw o e n z y m a tic
fo rm s . I t w a s n e c e s s a r y to d e te rm in e i f th e p h o to o x id a tio n w a s th e r e s u lt
■o f m e th y le n e b lu e s e n s i t i z a t i o n , o r th e r e s u l t o f th e 1, 1 0 -p h e n a n th ro lin e
z in c c o m p le x .
E x p e rim en ts w ere c a rrie d o u t in w h ic h s e n s i t i z a t i o n by
I ,.1 0 -p h e n a n th ro lin e w a s t e s t e d b y e x p o s in g th e en zy m e to lig h t in th e
p r e s e n c e o f OP ,but a b s e n c e o f m e th y le n e b l u e .
T h e se e x p e rim e n ts sh o w
t h a t 1, 1 0 -p h e n a n th ro lin e h a d no p h o to c h e m ic a l e f f e c t on th e e n z y m e , an d
d e m o n s tra te th a t th e lo s s o f p e p tid a s e a n d e s t e r a s e a c ti v ity is s o le ly th e
r e s u lt o f th e m e th y le n e b lu e s e n s i t i z a t i o n .
In o rd e r to c o m p le te ly
e lim in a te th e p o s s i b i l i t y o f m e ta l c h e la to r or c h e la te d c o m p le x InvoT vem e n t, a p o c a rb o x y p e p tid a s e -A y w a s p re p a re d b y th e m e th o d s o f V a lle e
on a S e p h a d e x G -2 5 c o lu m n .
(3)
'
The r e s u ltin g en zy m e w a s p h o to o x id iz e d
in th e p r e s e n c e o f m e th y le n e b lu e , a n d th e r e s u lts w ere fo u n d to be
c o m p le te ly a n a lo g o u s to th e p h o to o x id a tio n s in th e p r e s e n c e o f th e m e ta l
c h e la to r 1 ,1 0 -p h e n a n th ro lin e .
P h o to o x id a tio n o f c a rb o x y p e p tid a s e -A y in
th e p r e s e n c e o f th e s e c o n d c h e la to r , .EDTA, w a s in v e s t ig a te d , a n d w as
found to h a v e th e sa m e g e n e r a l e f f e c t on th e p e p tid a s e an d e s t e r a s e
a c t i v i t y , a s sh o w n in T ab le V.
It s h o u ld b e n o te d th a t th e in c u b a tio n o f
c a rb o x y p e p tid a s e -A y in th e p r e s e n c e o f EDTA r e s u lte d in a d e c r e a s e d
a b ility o f th e a p o e n z y m e to be r e a c tiv a te d w ith z in c , w h ic h w a s n o t
o b s e rv e d w ith I., 1 0 - p h e n a n th r o lin e .
No o th e r c h e la tin g a g e n ts w ere tr ie d .
S e v e ra l e n z y m e s h a v e b e e n p h o to o x id iz e d u s in g th e d y e R ose
B e n g a l. ^
^
W e s th e a d h a s d e s c r ib e d th e r e s u lts o f R ose B engal
s e n s i t i z e d p h o to o x id a t on on th e e n zy m e e n o la s e , a n d h a s in d ic a te d
■ (14)
th a t th e R ose B engal m ay be q u ite s p e c if ic fo r h is tid in e d e s t r u c t i o n .
T h e re fo re , in a n a tte m p t to e lim in a te so m e o f th e p o s s i b i l i t i e s o f r e s i ­
d u e s b e in g a tta c k e d in th e p h o to o x id a tio n
o f c a r b o x y p e p tid a s e - A y ,
-75-
R ose B engal w a s u s e d u n d e r th e sam e e x p e rim e n ta l c o n d itio n s a s th e
m e th y le n e b lu e .
The r e s u l t s o f F ig u re 15 c le a r ly in d ic a te th a t R ose Ben­
g a l ra p id ly d e c r e a s e s b o th th e p e p tid a s e a n d e s t e r a s e a c t i v i t y o f
c a r b o x y p e p tid a s e - A y .
In c o n tr a s t to th e m e th y le n e b lu e s e n s itiz e d
p h o to o x id a tio n , i t w a s found th a t R ose B engal d id n o t re q u ire th e
p r e s e n c e o f th e m e ta l c h e la to r .
The dye w a s e q u a lly e f f e c tiv e in r e ­
d u c in g th e p e p tid a s e a n d e s t e r a s e a c ti v ity o f th e n a tiv e e n z y m e , w h ic h
in d ic a te s th a t th e m e c h a n is m o f a c tio n o f th e d y e s a re d if f e r e n t.
This
d iff e r e n c e c o u ld be e x p la in e d in th e m a n n er in w h ic h th e y b in d to th e
p r o te in .
R ose B engal is a n a n io n ic co m pound o f a ro m a tic n a tu r e , and
c o n ta in s a fre e c a rb o x y l g r o u p . It s h o u ld b e n o te d th a t th e s u b s tr a te s
o f c a rb o x y p e p tid a s e a re lik e w is e a ro m a tic
g ro u p s
a n d c o n ta in fre e ' c a rb o x y l
T h u s , one m ig h t e x p e c t th a t R ose B engal c o u ld b in d to th e
en zy m e in a f a s h io n s im ila r to th a t o f th e s u b s t r a t e .
In th is c a s e , o ne
w o u ld e x p e c t th a t R ose B engal Would be c o n c e n tra te d m ore in th e a re a
o f th e a c tiv e c e n te r o f th e en zy m e th a n m e th y le n e b l u e , w h ic h is
c a ti o n ic .
The f a c t th a t m e th y le n e b lu e r e q u ire s a m e ta l c h e la to r c a n be
e x p la in e d in th e fo llo w in g m a n n e r. -M e th y len e b lu e , w h ic h is c a tio n ic ,
w o uld b e e x p e c te d to in te r a c t ,w ith a r e a s o f n e g a tiv e c h a rg e on th e
e n zy m e s u r f a c e .
T h u s , w h en th e z in c io n is p r e s e n t, th e d y e w ill be
a ttr a c t e d to a r e a s on th e e n zy m e s u rfa c e h a v in g a n e g a tiv e c h a rg e
d e n s it y .
S in c e th e en zy m e is n o t ra p id ly p h o to o x id iz e d u n d e r th e s e
c o n d itio n s , o n e c a n a s s u m e th a t t h e s e in te r a c tio n s i t e s , a n d th e s u b s e ­
q u e n t o x id a tio n s a t t h e s e s i t e s a re n o t c r itic a l to th e c a t a l y t i c a l a c ti v ity .
R em oval o f th e z in c , h o w e v e r, w h ic h is a p o s itiv e s p e c i e s , w ill e x p o se
a n e w n e g a tiv e ly c h a rg e d b in d in g s i t e .
T h is n ew s i t e , w h ic h is know n
to b e c a t a l y t i c a l l y f u n c tio n a l, w ill a ls o a t t r a c t th e m e th y le n e b lu e , an d
s u b s e q u e n t p h o to o x id a tio n a t th is s ite w o u ld be e x p e c te d to h a v e g ro s s
e f f e c ts on th e a c ti v ity o f th e e n z y m e . W h ile th e a b o v e e x p la n a tio n
s e e m s to s a t i s f a c t o r i l y e x p la in th e d a t a ,. a lte r n a te e x p la n a tio n s c a n a ls o
be g iv e n . .M e ta ls h a v e b e e n know n to in d u c e c o n fo rm a tio n a l c h a n g e s in
p r o te in s .
I t is p o s s ib le t h a t th e b in d in g o f z in c to th e a p o e n z y m e and
—76 —
% osteroce
activity remaining
M DIOP.Zn^
MB,0P,30minhv,Zn<4
RDjOfJ 30min hv, Z n4*
RB1SO m in hvr
j^ S ^ ^ S ^ ;- is ,a o r o ip i . ^ ^ ^ ' :
RB.OR Zn«
F ig u re 15. C o m p a riso n o f th e d y e s R ose B en g al a n d m e th y le n e b lu e a s
e f f e c tiv e s e n s i t i z e r s in th e p r e s e n c e a n d a b s e n c e o f 1, 1 0 - p h e n a n th r o lin e .
M e th y le n e b lu e an d R ose B engal c o n c e n tr a tio n s w e re 0 . 0 9 5 m g /m l.
P ro te in c o n c e n tr a tio n w a s 0 . 0 2 3 m g /m l. ZnClg w a s a d d e d w h e re in d i­
c a te d to r e v e r s e in a c tiv a tio n by 1, 1 0 - p h e n a n th r o lin e . The e s t e r a s e
a c ti v ity w a s d e te rm in e d a s d e s c r ib e d in " M e th o d s " .
-7 7 -
th e re m o v a l o f z in c from th e n a tiv e en zy m e a re a c c o m p a n ie d b y c h a n g e s
in c o n fo rm a tio n .
T h e se c h a n g e s m ig h t a llo w th e m e th y le n e b lu e to be
e f f e c tiv e on th e a p o e n z y m e , an d n o t on th e n a tiv e e n z y m e .
P re v io u s
s tu d ie s on c a r b o x y p e p tid a s e -A y h a v e s h o w n ; h o w e v e r, th a t re m o v a l o f
th e m e ta l p ro d u c e d a n a p o e n z y m e w h ic h c o u ld n o t be d is tin g u is h e d from
n a tiv e c a r b o x y p e p tid a s e -A y by a n y o f th e p h y s ic a l c r ite r ia W hich r e la te
(17)
to th e g ro s s c o n fig u ra tio n o f th e m o le c u le .
The h y p o th e s is o f th e b in d in g o f R ose B engal to th e a c tiv e s ite
c a n be t e s t e d in s e v e r a l w a y s . I t h a s b e e n d e m o n s tra te d p re v io u s ly
th a t p e p tid a s e a n d e s t e r a s e s u b s tr a te s form m e ta llo e n z y m e - s u b s tr a te
c o m p le x e s w h ic h p re v e n t th e d is s o c ia tio n o f th e m e ta l io n , a n d th e r e ­
fo re th e e x c h a n g e o f o n e m e ta l fo r a n o t h e r .^
^
In a d d itio n , th e
c o m p e titiv e in h ib ito r |3 -p h e n y lp ro p io n a te m a rk e d ly re ta r d s th e e x c h a n g e
o f ^ Z n ^ + <Z .n^+ a t th e a c tiv e s ite o f th e e n z y m e . ^ ^ T his p h en o m en o n
2_|_
2+
(2 0)
w a s a ls o sh o w n for th e Cd
a n d Co
c a r b o x y p e p tid a s e s . ' ; The
r e s u lts sh o w n in F ig u re 16 e s t a b l i s h th a t R ose B engal in te r f e r e s w ith th e
e x c h a n g e of z in c fo r c o p p e r in c o p p e r c a rb o x y p e p tid a s e .
The r e a c tiv a ­
tio n w a s a c c o m p lis h e d by th e a d d itio n o f 1 , 1 0 -p h e n a n th ro lin e to th e
in a c tiv e c o p p e r en zy m e s y s te m , in th e p r e s e n c e o f R ose B e n g a l,
fo llo w e d by th e a d d itio n o f z in c i o n s . As th e c o n c e n tra tio n o f R ose
B engal w a s i n c r e a s e d , th e a b ility to r e a c tiv a te th e e n zy m e w ith th e
a d d itio n o f z in c io n s w a s d e c r e a s e d , in d ic a tin g th a t th e R ose B engal
w a s , in e f f e c t , in te rfe rin g w ith e ith e r th e re b in d in g o f th e z in c io n , o r
th e r e l e a s e o f th e c o p p e r io n from th e e n z y m e .
The l a t t e r w o u ld be
c o n s is te n t w ith th e r e s u l t s o b s e rv e d b y V a lle e w ith th e s u b s tr a te HPLA
an d .p -p h e n y lp ro p io n a te .
S e c o n d ly , if R ose B engal is b in d in g a t th e
a c tiv e s i t e , i t s h o u ld be a c o m p e titiv e in h ib ito r o f th e e n z y m e .
The a b ility o f R ose B engal to fu n c tio n a s a c o m p e titiv e in h ib ito r
w a s i n v e s t ig a te d , an d th e r e s u lts a re sh o w n in F ig u re 17.
It a p p e a rs
th a t R ose B engal is in d e e d a c o m p e titiv e in h ib ito r o f c a rb o x y p e p tid a s e -A y .
As m e n tio n e d p r e v io u s ly , R ose B engal a n d th e s u b s tr a te s o f c a rb o x y p e p tid a s e a re a n io n ic .
T h u s , i t w o u ld n o t b e to o s u rp ris in g th a t R ose
—
jjl
78 —
RB a d d e d
F ig u re 16. The e f f e c t o f R ose B engal on th e e x c h a n g e o f C u
an d Zn
io n s in c a rb o x y p e p tid a s e -A y . P ro te in c o n c e n tra tio n w a s 0 .1 3 4 m g /m l
in a ll d e te r m in a tio n s . R e a c tiv a tio n o f th e in a c tiv e c o p p e r c a rb o x y p e p t i d a s e w a s a c c o m p lis h e d by in c u b a tin g th e c o p p e r e n z y m e , in th e
p r e s e n c e o f v a ry in g c o n c e n tr a tio n s o f R ose B engal b u ffe r, w ith 2 x 10
M , 1, 1 0 -p h e n a n th ro lin e an d th e n a d d in g a s u ita b le a liq u o t o f IO- ^ M
ZnCl2 b u ffe r. The R ose B engal b u ffe r u s e d h a d a c o n c e n tr a tio n of
3 m g /m l.
-79-
1300
1000
F ig u re 17. L in e w e a v e r-B u rk p lo t o f c a r b o x y p e p tid a s e a c t i v i t y in th e
p r e s e n c e a n d a b s e n c e o f R ose B e n g a l. R ose B engal c o n c e n tr a tio n
w a s 0 .0 0 1 m g /m l. P ro te in c o n c e n tra tio n w a s 0 . 0 0 5 4 m g /m l. The
e s t e r a s e a c ti v ity in th e p r e s e n c e o f R o se B e n g a l, ( o - o ) , a n d in th e
a b s e n c e o f R ose B engal ( o - o ) , w a s d e te rm in e d a t th e a p p ro p ria te
s u b s tr a te c o n c e n tr a tio n s a s d e s c r ib e d in " M e th o d s " .
—80 —
B engal w o u ld b in d a t th e a c tiv e c e n te r o f th e e n z y m e .
T his b in d in g is n o t
th e o n ly in s ta n c e o f a p p a r e n t a c tiv e s i t e b in d in g o f R ose B e n g a l.
S tu d ie s
on liv e r a lc o h o l d e h y d ro g e n a s e ., w h ic h a ls o b in d s a n io n ic s u b s t r a t e s ,
in d ic a te th a t R ose B engal is boun d to th e a c tiv e s ite o f th is en zy m e an d
f u n c tio n s a s w e ll a s a c o m p e titiv e in h ib ito r . ^
In a d d itio n , a n o th e r k in e tic s tu d y w a s u n d e rta k e n , in w h ic h th e
l o s s o f p e p tid a s e a n d e s t e r a s e a c t i v i t i e s w e re p lo tte d a s a fu n c tio n o f
tim e .
A s e m i-lo g a rith m ic p lo t o f th e s e d a ta g iv e s th e c u rv e in F igure
18 w ith tw o in d e p e n d e n t s l o p e s . .The f a c t t h a t s u c h a k in e tic cu rv e
w a s o b ta in e d m ig h t in d ic a te th a t m ore th a n o n e r e a c tio n is o c c u rrin g .
The i n itia l m ore ra p id r e a c tio n c a n b e a s s o c i a t e d w ith th e i n i t i a l lo s s
o f th e p e p tid a s e a n d e s t e r a s e a c t i v i t y , th e r e s u lt o f p h o to o x id a tio n o f
th e a c tiv e s i t e . .The s lo w e r r e a c tio n c a n b e r a tio n a liz e d .
I t is lik e ly
th a t th e in i t i a l o x id a tio n o c c u rs a t th e a c tiv e s ite o f th e e n zy m e and
m a n ife s ts i t s e l f in th e lo s s o f p e p tid a s e an d e s t e r a s e a c t i v i t y .
If
th e o x id iz e d r e s id u e h a d in a d d itio n to i t s c a ta ly tic fu n c tio n , a
s tr u c tu r a l r o l e , o r if o x id a tio n o f a r e s id u e in th e a c tiv e s i t e e x p o s e d
to o x id a tio n a s e c o n d r e s id u e o f s tr u c tu r a l im p o rta n c e , th e te r tia r y
s tru c tu re o f th e e n zy m e w o u ld be a l t e r e d . As th e d e n a tu r a tio n o f th e
p ro te in m o le c u le s p ro g r e s s e d th e re w o u ld be m ore an d m ore p h o to o x id iz a b le r e s id u e s e x p o s e d , an d t h e s e w o u ld c o m p ete fo r th e dye
m o le c u le s an d s in g le t o x y g en , th u s c a u s in g a n a p p a r e n t d e c r e a s e in
r a te o f l o s s o f p e p tid a s e a n d e s t e r a s e a c ti v ity w ith tim e .
The d y e , c r y s ta l v i o l e t , h a s b e e n sh o w n to b e s p e c if ic fo r th e
( 21)
p h o to o x id a tio n o f c y s te in e r e s i d u e s .
' T h e re fo re , c r y s ta l v io le t
s e n s i t i z e d p h o to o x id a tio n o f th e c a r b o x y p e p tid a s e -A y w a s u n d e rta k e n ,
an d i t w a s found th a t th e en zy m e w a s n o t a ffe c te d u n d e r t h e s e c o n d i­
t i o n s , e v e n in th e p r e s e n c e o f th e c h e la to r , 1 ,1 0 - p h e n a n th r o lin e .
The p h o to o x id a tio n o f h is tid in e h a s b e e n sh o w n to b e p H d e p e n d e n t . The r e la tiv e r a te s o f o x id a tio n o f h is tid in e a s a
fu n c tio n o f pH w a s s ig m o id a l, a n d h a d a n in f le c tio n n e a r pH 7 . 3 .
am ino a c id s tr y p to p h a n , ty r o s in e , a n d m e th io n in e , w h ic h a re a ls o
The
-81-
60
tim e
sec
F ig u re 18. S e m i-lo g a rith m ic p lo t o f l o s s o f e s t e r a s e a c ti v ity a s a
fu n c tio n o f tim e . P ro te in c o n c e n tra tio n w a s 0 . 0 2 8 m g /m l. R ose
B engal c o n c e n tr a tio n w as 0.10 m g /m l. P h o to o x id a tio n s w e re
c o n d u c te d in 0 .1 M T ris b u ffe r, pH 7 .5 a t 3 ° C . , c o n ta in in g 1.0 M
N a C l. 500 w a tt lig h t s o u rc e w a s fo c u s e d th ro u g h a n f 1 .6 l e n s .
S a m p le s w e re 4 .3 cm from th e l e n s . The e s t e r a s e a c t i v i t y w a s
d e te rm in e d a s d e s c r ib e d in " M e th o d s " .
—82 —
s u s c e p tib le to p h o to o x id a tio n do n o t sh o w th is pH d e p e n d e n c e „
S ig n if ic a n tly , w h e n c a r b o x y p e p tid a s e - A y is p h o to o x id iz e d in th e p r e ­
s e n c e o f R ose B e n g a l, o r in th e m e th y le n e b lu e 1 , 1 0 -p h e n a n th ro lin e
s y s te m , (F ig u re 19), a sig m o id a l pH p ro file is o b ta in e d w h ic h is a lm o s t
id e n tic a l to th a t e x h ib ite d fo r th e en zy m e e n o l a s e : ^ 4^ T h is pH d e ­
p e n d e n c e o f th e p h o to o x id a tio n o f c a rb o x y p e p tid a s e -A y s u g g e s ts th a t
th e r a te lim itin g s te p is c o n tro lle d b y th e o x id a tio n o f a h is tid in e
re s id u e .
If s u b s tr a te o r c o m p e titiv e in h ib ito r s w ere to p r o te c t th e
e n zy m e from p h o to o x id a tio n , s u b s ta n tia l e v id e n c e th a t th e in itia l
o x id a tio n w a s o c c u rrin g a t th e a c tiv e s i t e o f th e e n z y m e w o u ld be o b ­
ta in e d .
T h u s , s tu d ie s w e re u n d e rta k e n to d e te rm in e if th e s u b s tr a te
w o u ld p r o te c t.
The s u b s tr a te c h o s e n w a s h ip p u r y l- L - p h e n y la la n in e ,
th e p e p tid a s e s u b s t r a t e .
S u b s ta n tia l s u b s tr a te p ro te c tio n w a s
o b s e rv e d in th e R ose B engal s y s te m (F ig u re 20) .
The c o m p e titiv e
in h ib ito r , .(3-phenylpropionate w a s a ls o tr ie d a s a p ro te c to r a g a in s t
th e p h o to o x id a tio n . .P re v io u s s tu d ie s h a v e sh o w n th a t g -p h e n y lp ro ( 2 2 23)
p io n a te b in d s to th e a c tiv e s ite o f th e e n z y m e .
'
'
It is o b v io u s
from th e d a ta in F ig u re 21 th a t-(B -p h e n y lp ro p io n a te d o e s p r o te c t th e
e n zy m e a g a in s t p h o to o x id a tio n .
L ik e w is e , .X -ray s tu d ie s h a v e show n
th a t g ly c y lty r o s in e , a s u b s tr a te o f th e e n z y m e , b in d s o n ly a t th e
m e ta l b in d in g s i t e ;
T h e re fo re , if th e p h o to o x id a tio n w e re o c c u rrin g
on th e s u rfa c e o f th e en zy m e a t ra n d o m , o n e w o u ld n o t e x p e c t
g ly c y lty ro s in e to p r o t e c t .
G ly c y lty ro s in e s u b s tr a te p ro te c tio n w as
u n d e r ta k e n , a n d i t w a s found to s ig n if ic a n tly h in d e r-th e p h o to o x id a tio n
o f th e en zy m e (F ig u re 22) .
T his r e s u l t e s t a b l i s h e s th a t th e p h o to -
o x id a tio n le a d in g to in a c tiv a tio n o f .the en zy m e is .o c c u rrin g a t th e
a c tiv e s i t e o f th e e n z y m e .
I t w a s o f i n t e r e s t to d e te rm in e i f a n y g ro s s
c o n fo rm a tio n a l c h a n g e s o c c u rre d s u b s e q u e n t to th e p h o to o x id a tio n o f
c a r b o x y p e p tid a s e . I t w a s f e lt th a t t h e s e c o n fo rm a tio n a l c h a n g e s m ight
be d e te c te d th ro u g h S e p h a d e x c h ro m a to g ra p h y . .A s e r ie s o f e x p e rim e n ts
w e re u n d e rta k e n w h e re c a r b o x y p e p tid a s e w a s p h o to o x id iz e d w ith R ose
-83 —
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F igure 19. The pH d e p e n d e n c e o f th e m e th y le n e b lu e - 1 ,1 0 - p h e n a n th ro lin e
s e n s i t i z e d p h o to o x id a tio n (o -o ) an d th e R o se B engal s e n s i t i z e d p h o to ­
o x id a tio n (o -o ) a re s h o w n . P ro te in c o n c e n tr a tio n w a s 0 . 0 2 m g /m l.
The c o n c e n tr a tio n s o f m e th y le n e b lu e a n d R o se B engal w e re 0.19 m g /m l
a n d 0 .1 0 m g /m l r e s p e c t i v e l y .
—84 —
% activity
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s u b * p -3 1+ ^ 0 p / / / / / M
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% % % %
610 s u b v S m i n h v
F ig u re 2 0 . P ro te c tio n a g a i n s t R ose B engal p h o to o x id a tio n o f
c a rb o x y p e p t i d a s e -A y w ith th e s u b s tr a te h ip p u r y l- L - p h e n y la la n in e .
H ip p u ry l- L -p h e n y la la n in e c o n c e n tra tio n w a s 10~2 M . R ose B engal
c o n c e n tr a tio n w a s 0 . 0 4 m g /m l a n d th e p ro te in c o n c e n tr a tio n w as
0 . 0 4 m g /m l. P e p tid a s e a c ti v ity w a s d e te rm in e d by re m o v in g a
10 \ X a liq u o t a n d a s s a y in g in th e n o rm al m a n n e r.
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F ig u re 21. P ro te c tio n a g a in s t R ose B en g al p h o to o x id a tio n w ith
g - p h e n y lp r o p io n a te . R ose B engal c o n c e n tr a tio n w a s 0 . 0 3 m g /m l.
g -p h e n y lp ro p io n a te c o n c e n tr a tio n w a s 2 x 10~2 m . P ro te in c o n c e n ­
tr a tio n w a s 0 . 0 8 m g /m l. The e s t e r a s e a c ti v ity w a s d e te rm in e d a s
d e s c r ib e d in " M e th o d s " .
—85 —
% peptidase
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50
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S t K S S S S K S C 50 "=In hr
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F ig u re 2 2 . P r o te c tio n a g a in s t R ose B en g al p h o to o x id a tio n w ith
g ly c y lty r o s in e . F in a l c o n c e n tr a tio n s o f c o m p o n e n ts w e re a s fo llo w s:
R ose B e n g a l, 0 . 0 3 m g /m l; c a r b o x y p e p tid a s e , 0 . 0 8 m g /m l; g ly c y l­
ty r o s in e , 0. 001 M. The p e p tid a s e a c ti v ity w a s d e te rm in e d a s
d e s c r ib e d in “ M e th o d s " .
-8 6 -
B e n g a l, an d th e n p la c e d on a G -7 5 c o lu m n .
The e lu tio n p a tte r n s in th e
e x p e rim e n ta l s e c tio n (F ig u re s 1 , 2 , a n d 3 ), in d ic a te th a t a s p h o to o x i­
d a tio n tim e i n c r e a s e s , a ra p id ly m oving p e a k a p p e a rs in th e e lu tio n
p a tte r n o f th e e n z y m e , w h ic h s u g g e s ts t h a t p o s s ib ly so m e a g g re g a tio n
is o c c u r rin g .
T his a g g re g a tio n w a s s u b s e q u e n tly co n firm e d w h en it
w a s found th a t upon p ro lo n g e d p h o to o x id a tio n th e p ro te in a c tu a lly
p r e c ip ita te d from s o lu tio n . .T his p r e c ip ita te w a s c o lle c te d b y c e n tr if u ­
g a tio n , w a s h e d w ith w a te r , a n d th e n s u b je c te d to am in o a c id a n a l y s i s ,
a n d no h is tid in e c o u ld be d e te c te d in th e s a m p le s .
C h ro m a to g ra p h y on
TEAE c e ll u lo s e p ro d u c e d a v e ry in te r e s tin g an d so m e w h a t s u rp ris in g
r e s u l t . .W hen th e u n o x id iz e d en zy m e w a s p a s s e d th ro u g h th e TEAE
colum n., fiv e p e a k s w e re o b s e r v e d in th e e lu e n t p a tte r n . W h e n fr a c tio n s
w e re c o lle c te d a s th e v a rio u s p e a k s e m erg ed from th e c o lu m n , an d th e
p e p tid a s e a c ti v ity d e te rm in e d , i t w a s found th a t e a c h p e a k c o n ta in e d
e n z y m a tic a c t i v i t y .
C a rb o x y p e p tid a s e -A y a p p a r e n tly e x i s t s in a t l e a s t
fiv e a c tiv e c o n fo rm a tio n s a s i t is p u r c h a s e d , or a s p re p a re d b y th e
m eth o d o f A n so n .
T h e se v a rio u s form s h a v e lik e w is e b e e n o b s e rv e d by
D r. ,N e u rath a n d c o w o rk e rs a t th e U n iv e rs ity o f W a s h in g to n , w ho h av e
b e e n a b le to s e p a r a te t h e s e su b fo rm s b y c h ro m a to g ra p h y o n DEAE
c e ll u lo s e in th e p r e s e n c e o f th e in h ib ito r p -p h e n y lp ro p io n a te . ^ ^
It
a p p e a rs th a t TEAE is c a p a b le o f p a r tia l s e p a r a tio n o f t h e s e form s
w ith o u t th e p r e s e n c e o f th e in h ib ito r .
If th e p h o to o x id iz e d en zy m e
is p la c e d on th e TEAE c o lu m n , a s im ila r p a tte r n is o b s e r v e d , a s show n
in F ig u re s 4 , .5, an d 6 in th e e x p e rim e n ta l s e c tio n .
O ne s h o u ld n o te ,
h o w e v e r, th a t a s p h o to o x id a tio n tim e i n c r e a s e s , th e a m o u n t o f en zy m e
e lu te d from th e co lu m n is d e c r e a s e d , b u t th e r e is no c h a n g e in th e
a c ti v ity p ro file o f th e e lu tio n p a t t e r n , w h ic h s u g g e s ts th a t th e p h o to o x id iz e d en zy m e is tig h tly bou n d to th e co lu m n an d is n o t e lu te d .
A ttem p ts w e re m ade to e lu te th e o x id iz e d en zy m e from th e TEAE colum n
by in c r e a s in g th e s a l t c o n c e n tra tio n o f th e e lu e n t.
The so d iu m c h lo rid e
c o n c e n tra tio n w a s in c r e a s e d to fo u r m o la r, w ith o u t e lu tio n o f p ro te in
from th e c o lu m n .
S in c e h ig h s a l t c o n c e n tr a tio n s fa ile d to e lu te th e
—
87 —
o x id iz e d en zy m e from th e c o lu m n , i t w a s c o n c lu d e d th a t ir r e v e r s ib le
a g g re g a tio n h a d o c c u rre d fo llo w in g th e p h o to o x id a tio n ..
No fu rth e r
a tte m p ts w e re m ade to u s e io n e x c h a n g e co lu m n s to s e p a r a te th e
o x id iz e d en zy m e from u n o x id iz e d e n z y m e .
P h o to o x id iz e d en zy m e
w a s s u b je c te d to S e p h a d e x G -7 5 colum n c h ro m a to g ra p h y , a n d th e
r e s u l t s s u p p o rt th e h y p o th e s is th a t p h o to o x id a tio n is le a d in g to
a g g r e g a tio n .
F ig u re s 8 , - 9 , a n d 10 o f th e e x p e rim e n ta l s e c tio n sh o w
th e e lu tio n p a tte r n s o f p h o to o x id iz e d e n zy m e on S e p h a d e x G - 7 5 .
The
f a s t ru n n in g p e a k a p p e a rs w ith p h o to o x id a tio n , an d in c r e a s e s in in te n ­
s ity w ith in c r e a s e d tim e o f p h o to o x id a tio n .
O ne c o n c lu d e s from th is
th a t p h o to o x id a tio n d o e s r e s u lt in a g g re g a tio n o f th e e n zy m e m o le c u le s .
T his a g g re g a tio n m ay r e s u l t by s e v e r a l m e c h a n is m s .
The p h o to o x id a tio n
o f th e n a tiv e p ro te in m ay a lte r th e s u rfa c e c h a rg e d is tr ib u tio n so th a t
e l e c t r o s t a t i c in te r a c tio n le a d s to a g g r e g a tio n .
If th e a g g re g a tio n is of
th is ty p e , th e c h a rg e in te r a c tio n s m u st b e e x tre m e ly s tr o n g , s in c e 4 M
N a C l d o e s n o t b re a k i t up..
A s e c o n d p o s s i b i l i t y is th a t p h o to o x id a tio n
le a d s to c o n fo rm a tio n a l c h a n g e o f th e n a tiv e m o le c u le , a n d th a t th e s e
co n fo rm e rs th e n a g g r e g a te .
T his p o s s i b ility is c o n s is te n t w ith th e ra te
d a t a , th e am ino a c id a n a ly s is d a ta , an d th e s e d im e n ta tio n s tu d y .
As
p re v io u s ly m en tio n ed ., tw o d is tin c t r a te s a re o b s e rv e d (F ig u re 18),
T h e se r a te s c o u ld c o rre s p o n d to (a) th e r a te o f o x id a tio n o f n a tiv e
e n z y m e , a n d (b) p h o to o x id a tio n o f n e w ly form ed c o n fo rm e rs . Amino
a c id a n a ly s is o f th is a g g re g a te d m a te r ia l, c o lle c te d by p r e c ip ita tio n ,
sh o w s th a t a ll th e h is tid in e h a s b e e n d e s tr o y e d .
It is V ery te m p tin g
to s u g g e s t t h a t th e c o m p le te d e s tr u c tio n o f th e re m a in in g h is tid in e s is
o c c u rrin g a f te r th e n e w ly form ed c o n fo rm e rs a re p ro d u c e d .
If c o n fo r­
m a tio n a l c h a n g e s a re o c c u r rin g , th is w o u ld b e th e f ir s t s u c h c h a n g e
o b s e rv e d in a n e n z y m a tic s y s te m w h ic h w a s p h o to o x id iz e d .
The e f f e c ts o f v a rio u s o th e r m e ta l io n s s u b s e q u e n t to p h o to ­
o x id a tio n o f th e en zy m e w e re in v e s t ig a te d .
It h a s b e e n sh o w n th a t if
th e n a tiv e z in c en zy m e is in c u b a te d w ith o r th o - p h e n a n th r o lin e , a n d
th e s y s te m th e n flo o d e d w ith c o b a lt io n , th e a c tiv ity is in c r e a s e d
-8 8 -
ro u g h ly t w o - f o ld from t h a t o f th e n a t i v e e n z y m e . If th e e n z y m e is
p h o to o x id iz e d fo llo w in g th e i n c u b a t io n w ith 1, 1 0 - p h e n a n t h r o l i n e , a n d
th e n c o b a l t i s a d d e d to th e s y s t e m , th e a c t i v i t y is d e c r e a s e d p r o ­
p o r t i o n a t e l y w ith th e tim e o f p h o to o x id a tio n a s w a s o b s e r v e d w ith z i n c .
In o th e r w o r d s , i f it i s o b s e r v e d t h a t in a c e r t a i n p e rio d o f tim e fifty
p e r c e n t o f th e a c t i v i t y w a s l o s t , w ith th e r e a d d itio n o f z i n c , i t w ould
a l s o b e o b s e r v e d t h a t fif ty p e r c e n t o f th e a c t i v i t y is l o s t on th e
a d d itio n o f c o b a l t .
This i n d i c a t e s t h a t th e p h o to o x id a tio n n o t o n ly
d e s t r o y s th e c a p a c i t y o f z in c to r e a c t i v a t e th e e n z y m e , b u t a l s o th e
c a p a c i t y o f c o b a l t to r e a c t i v a t e th e e n z y m e .
A c o b a l t c a r b o x y p e p tid a s e
(2 5)
w a s p re p a r e d b y d i a l y s i s o f th e z in c e n z y m e a g a i n s t a c o b a l t s o lu tio n .
This c o b a l t e n z y m e w a s p h o to o x id iz e d a n d found to h a v e e s s e n t i a l l y
th e s a m e p r o p e r tie s a s t h a t o f th e z i n c e n z y m e .
That i s , p h o to o x id a tio n
o f th e c o b a l t e n z y m e l e a d s to th e d e s t r u c t i o n of b o th p e p t i d a s e and
e ste ra se a c tiv itie s.
O th e r m e ta llo e n z y m e s w e re p re p a re d a s d e s c r ib e d
in th e e x p e r im e n ta l s e c t i o n , a n d t h e i r p r o p e r tie s s u b s e q u e n t to p h o to o x i­
d a tio n d e te r m in e d .
The r e s u l t s o f t h e s e p h o to o x id a tio n s w e re s im ila r to
t h o s e o b ta in e d w ith th e z in c e n z y m e .
T h e s e e x p e r im e n ts s u p p o rt th e
h y p o t h e s i s t h a t th e a c t i v a t i n g m e ta l io n s b in d a t th e s a m e s i t e in th e
enzym e.
The r a te o f p h o to o x id a tio n s e e m e d to d e p e n d to som e e x te n t
u p o n th e b u ffe r s y s te m e m p lo y e d d u rin g th e p h o to o x id a tio n .
It w o u ld
b e i n t e r e s t i n g to fu rth e r i n v e s t i g a t e th e e f f e c t of v a r io u s b u ffe rs on th e
p h o to o x id a tio n of o th e r e n z y m e s a s . w e l l a s c a r b o x y p e p tid a s e - A y .
I
h a v e o b s e r v e d t h a t th e r a t e o f p h o to o x id a tio n c h a n g e s w ith d if f e r e n t
b u ffe r s y s t e m s , b u t t h e g e n e r a l a p p e a r a n c e o f th e s e m i - l o g r a t e p lo t
re m a in s th e s a m e .
It is p o s s i b l e t h a t th e d iff e r in g r a t e s m ay r e f l e c t
m inor c h a n g e s in th e c o n fo rm a tio n o f th e t o t a l p ro te in m o l e c u l e .
.A t y p i c a l am ino a c i d a n a l y s i s s u b s e q u e n t to p h o to o x id a tio n is
sh o w n in F ig u re 2 3 .
The m o s t im p o rta n t a s p e c t o f t h i s a n a l y s i s i s th e
a lm o s t t o t a l d e s t r u c t i o n o f h i s t i d i n e in p h o to o x id iz e d m o l e c u l e s .
H isti­
d in e i s th e p r in c ip a l am in o a c i d a f f e c t e d , t h e a n a l y s i s sh o w in g l i t t l e
l o s s o f a n y o th e r am ino a c i d .
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F ig u re 2 3 . A t y p i c a l a m in o a c i d a n a l y s i s o f o x id iz e d (p. m ole**) and
u n o x id iz e d (p m ole*) c a r b o x y p e p tid a s e - A y . The l o s s of c y s t e i n e w a s
v a r i a b l e d e p e n d in g on th e o x y g e n c o n t e n t o f th e h y d r o ly s is m ix tu re .
—9 O~
The p h o to o x id a tio n o f c a r b o x y p e p t i d a s e -A § i s w o rth y o f fu rth e r
(3)
c o m m e n t.
This p h o to o x id a tio n r e s u l t s in i n c r e a s e d e s t e r a s e a c t i v i t y ,
a n d d e c r e a s e d p e p t i d a s e a c t i v i t y . .At l e a s t tw o e x p l a n a t i o n s c a n be
g iv e n for t h i s a p p a r e n t d i s c r e p a n c y . .E ith er th e c o n fo rm a tio n o f th e
a p o - § form in th e z in c b in d in g s i t e i s s u c h t h a t i t in h i b i t s th e d y e
b in d in g , or th e m e ta l b in d in g s i t e d o e s n o t in v o lv e h i s t i d i n e .
SUM M ARY
It w a s found t h a t d y e - s e n s i t i z e d p h o to o x id a tio n u s in g e ith e r
m e th y le n e b lu e or R ose B engal a s s e n s i t i z e r s r a p id ly d i m in is h e s both
th e p e p t i d a s e a n d e s t e r a s e a c t i v i t i e s o f c a r b o x y p e p tid a s e - A y . It is
s i g n i f i c a n t th a t b o th a c t i v i t i e s a re s im u l t a n e o u s l y d im in is h e d by th is
c h e m i c a l m o d if ic a tio n p r o c e d u r e , s i n c e h e re to fo re a d u a l i n a c t i v a t i o n
h a s b e e n a c c o m p lis h e d o n ly b y re m o v a l o f th e m e ta l from th e e n z y m e .
All o th e r c h e m ic a l m o d if ic a tio n s e m p lo y e d on c a r b o x y p e p tid a s e - A
p r e v i o u s l y r e p o r t e d , in c lu d in g m e th y le n e b lu e s e n s i t i z e d p h o to o x id a ­
tio n o f c a r b o x y p e p tid a s e - A § h a v e r e s u l t e d in a n i n c r e a s e in e s t e r a s e
a c t i v i t y a n d a d e c r e a s e in p e p t i d a s e a c t i v i t y .
A d if f e r e n c e w a s found i n th e m e c h a n is m o f a c t i o n b e tw e e n
R ose B engal a n d m e th y le n e b l u e .
R ose B engal f u n c tio n s a s a v e ry
e f f e c t i v e s e n s i t i z e r for b o th c a r b o x y p e p tid a s e - A y a n d a p o c a r b o x y p e p t i d a s e - A y , w h ile m e th y le n e b lu e s e n s i t i z e s th e p h o to o x id a tio n o f o n ly
th e m e t a l - f r e e form o f .th e enzyme..
Both s e n s i t i z e r s s h o w a m arked
pH d e p e n d e n c e a n d g iv e i d e n t i c a l s ig m o id a l c u rv e s w h ic h a re t y p i c a l
o f th e pH d e p e n d e n c y o f th e d y e - s e n s i t i z e d p h o to o x id a tio n o f th e
im id a z o le m o ie ty .
C r y s t a l v i o l e t , a s e n s i t i z e r w h ic h i s r e p o r te d to be
s p e c i f i c for c y s t e i n e r e s i d u e s , w a s found to be c o m p le te ly i n e f f e c tiv e
on a p o c a r b o x y p e p tid a s e - A y .
Amino a c i d a n a l y s i s .of p h o to o x id iz e d e n zy m e r e v e a l e d th e l o s s
o f o n ly h i s t i d i n e r e s i d u e s .
P h o t o s e n s i t i v e am ino a c i d s s u c h a s
c y s t e i n e , t y r o s i n e , a n d m e th io n in e re m a in e d e s s e n t i a l l y u n a l t e r e d .
S ig n if ic a n t p r o te c tio n a g a i n s t t h i s p h o to o x id a tio n w a s d e m o n s tr a te d
u s in g th e s u b s t r a t e s h i p p u r y l - L - p h e n y l a l a n i n e , g l y c y l t y r o s i n e , a n d th e •
c o m p e titiv e in h ib ito r p - p h e n y lp r o p io n a te .
K in etic s t u d i e s i n d i c a t e t h a t
R ose B engal a l s o f u n c tio n s a s a c o m p e titiv e in h ib ito r o f c a r b o x y p e p ti­
d a s e - A y a n d p r e v e n ts th e e x c h a n g e o f m e ta l io n s a t th e a c t i v e c e n te r
o f th e e n z y m e .
The p h o t o l a b i l i t y o f s e v e r a l m e ta llo d e r i v a t i v e s of
c a r b o x y p e p tid a s e - A y w e re i n v e s t i g a t e d .
C o n fo rm a tio n a l c h a n g e s s u b ­
s e q u e n t to p h o to o x id a tio n a n d th e p h o t o l a b i l i t y o f th e e n z y m e in v a rio u s
b u ffe r s y s t e m s w e re a l s o i n v e s t i g a t e d .
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Ilinn mu
MONTANA STATE UNIVERSITY LIBRARIES
3 1762 10010415 5
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