Ion Excba nge CbrC'lm& togrE.phy ftudy
On The
Isol~tion
~eaminsse
1f
~denosine
Prom Prog Liver
by C&ry L. Thrsll
under Dr. Pane-Yai :,'Ta
-sub8itted in fulfillment of the
f:enior H ,ncrs Thesis Pr,1ject, ID 499
'fTay
19, 19'71
';'
!
I r8C0!'1f11end
~r0gr2m
of
thi~
~~ll
th"?::;is fDT'
~tnte
~:ccent;:nce
U~iv"?rsity
for
by the Honors
g!'~~u8tion
pith
(':'besis /,dvis)r)
Dep&rt~ent
d&te
of
Che~istry
n
~he
I~~lati~r
~f
:.de~~sire
)ccurs
If t.r
~ ~I
d.
,,'l'y-nr
,1
< r-.
'
J.
, . .J.. C"1. ....:.,.
..:: Y-'
..L ~ _ '-) 1
r
I.e.. t.~
:,,~;
'-, L_
t.._........
i·-'(":._ ,-.
")
.'~,
I' _
y
..
..::.' ", ~-)
'"'
-.
i
':.1ined.
)~ojrct.
: tnJcturc:
tJ:·ic
t:)
f~Te~t
livi~:
systS?s.
.~
CE.1:
J~~
i..::~ 'ctivit;; 1
'18.1'Y
c·
i'cr: tis S1;:8."~
c'"
'-'. 2)
r- Cl' PS
,,',...
p::: TticlJl~.l"
("::]
..... r'~l./,
1\
\_-'
..ll._,
.... --:
Cll.i.'_~
1
h."
• ". '\ '
",
":"'''-''~J-~,I,...,'I....),\___ ,...;,.j
-,.".,~,)
(,
\J.-'.
L".).o.-."
...
r'
C!.~':::
-:'
"~-,
t',f:r~ .
~ "',
..L1
, ,;'
t-;IJ
f' ~:J ,-,
.. c.:)l :'.-YJI'l
(1965)
2""
.':.a. ,
""ONI
<~d-d
r")'-
I
-
,_.
_,
J. >:.,
V
....
(In''E')
o:JO I •
':>:-1 s~ ~;r , J. -" ., -':a ';.
~.:..~Y}:Y[)ys.
16,
'-,,)7
l .:q~8-<-'"
L.
~~.,
(10"~)
".\:)v.
. .,
J
-,
.
ver
4
It
b~s
been roticed tnat the
r&ti~~
of
t~~
~~o~nt
of
iif:~re~t
aT":
latl~n
to
t~is
[,:.11 ;-,',.'(>"5. b,/ 1
8.:n
0nzy~e.
r~
C(11l;,]r
"
CO~pOSl
a~lno
5,.....
"Ul" !'f·:·... ,
,c,:=;
574-534 (lH6(3).
C"
r;xcr;;:lr:e
'
sc i Q
chr::::·iF, togT'(~>0y. c
It;:;.:::
~'·~;lJrr1
. t'18n
,
!;.,.,
5
cc~p~siti~n
is
k~own
for a form
on
OJ.
D~vlng
ct
nolecu1ar wei -ht of
~p1 ~l'lt~~t~0n
6
1... ~r)lu'~r
.._\. ____
'';-10(;
r
J
_'
... _
1_ ... ~.
....... ...
1
•
(Ta.ble I)
T !~. .3LE. I
t'fOEidues
t.E' j:';: rta. t e
14
15
'I'hr"'orine
Cerine
Glutam&te
Pr,)line
Crl:lCi2:1C
A1anin rc.
Cystein"=:
Valim;
I ~- ~)lf:l:ci.r:e
16
14
16
,;i~'ti'-lini-~
started
~.
L-'
:}c::lac t·Jsa;,-,inc
1
·~luc:_,sal;.ine
9
~~s
6
:'!'y:}L; ;;han
5
19
~roj0ct
This
Le"Jcine
'Y.'yrosinp
?heny1alan.ine
Lysine
.'-,rgi. ine
:-Ethi :::'Y} i~-le
~s
a learning exp'r': (~-~ce
,J :'.' "3
::;i f' f i c'Ll t i
6 Pf """'-r:r''''
':S
1.,
~
J.I.
I
_:~
,,~
-' •
~)
Cl't
•
f-;
S
..
-
v[18
en r <):, E•. t
J-
6
tissu~,
centrif~gati0~
to rs
av~
tissue
Lt
;:;:rj
at
~~60nm
a:~air:st
8.
~&teri&l,
c~ch
W?.ter blar::.
1'"cleic
step
sblt
~r~cti0~-
e~7!~e
2ctiv~ty
7
v'Lcit/, s'Eocific act::vity,
~,
.~-'
,
'
c::...l.. ......
tGta1 8.ctlvity.
~
<'11 \c"c:tr"
rl};,T "-"'
..
ct tC"
t';'
~-;
~~*.
/~'·l'r.
_.a._.
T~te.l\ctivity
(T;Jt. E:Ct.)
ct,~·ck
:!,! G.- • /l"J~
(~:
:
~~:
~;ill)te:
5. C __'li;
'~nlJs~qha t€
(l.l~(j.
10 :::1/10(; :-21
This was kept
frG2e~
Ir:. tne fr::ls
8rior
t~
use.
r.
i·.)T'·"~
;::1.8,
8
t~~~6ratures
is
-
:)f the
~
Dr"
\ ..,
.
~tis
for all apsrati8rs
is necessary to preserve
enzyme activity.)
seiV8.
~(r:)te:r:s
:.mly st hL;h satura.tion.
ce~tr~tio~s
cne can
iS01~te
Thus by findir,; t:1E rL;lJ:
the en7yme of interest with
aration, how€,ver.
r::irjed
~'r]"J
a given t; ble.
~roteins
l~l;J
T~e::)
~o~
.,......
fractiDr: was
7"')
;~.,
... ,
...
,~
CO!}-
ir:cre-
~; ~~ by a~~ing
9
ste~.
After
SG~e
cO~Ei~0ratio~,
it
~~s
~scii8d
that
a~
ion ex-
ussd.•
;:cls~J
Hel buffer at pE 8.6.
er-zyrne ':.'as
8.S
say
result~;
sr'.-: as follc)'·'Js:
Phosphate (JUffer
;)rot. CClnc.
Vel.
E.r.'. act 4~
Tot. act.
9~~.
chra~&tograph!
6
D'; prot ./ml
)l7.,! .~~d. /r::in.
49.0
21. :1
1, ~160
The resDlts indicate
neN
':'ris buffer
~)
7 u~ ~i./~g prat.-min.
,<:1:) U!I::j:TJi D.
~:;J.
~:
advs~se
effect.
In fact, it seems that
rneth0d ta be tried.
; suspensiOn of
55~ fractio~s
was m2de in 0.U5
~ ~ris
buffer
10
the following assay:
su'oern8.tant
?r)t. C-,nc.
Vel.
S:,.
act.
T:)t. act.
pellet- activity
it :Dust Le
',vi to
t~e
used.
.rUal~r2G1.
negli~ible
to
chrevatclgr,s.ohy.
A 60 sl
fractio~
'T.'i,is is
r~ot
','{l~ 16-, I12.Y
inter fere
r.eces s;:,ry "Ii t!1 ,:el fil trc ti:)D.
resultpd.
Fr :;; c t
'--.'
a~proxi~8tely
t?xces S sc.lt
rer:DVA
l' , I -, , c:
~.
.... .1.""".
(~
,.....
1.7 rnl/nin. (Fi;ure 1)
collected * a~:d
::t 1 )'
_
~hrihka~e
of the bed
-~E
the fractiom, "'Ere as follov:s,:
*
A
Gils~n
automatic frsctiar. collector
used 11'2
2
cold box
11
fra c tion
0
58
5q
60
-
:i/
vel. (~0.D. x .0025 Abs.
)
IJin.
~5 pI sa;rn.
56
0
91
.75
q"
.67
';.:
00:;
.68
.08
;~)4
.70
0
• Z'
95
.17
,; E;
.,:;:.,v
'-'
... k
.<-.:
61
-
64
84
'g""
v
~"J
:37
•
5~
v
97
.56
..::',7
33
39
.67
98
.42
99
.5
90
.75
~6
.68
'-~
The results 'vere as [011y·"s:
fr~ct.
vel. (sa~e)
10:J
101
3.42
1. ~:~2,
102
1.~:5
104
IJ3 - 1;:"8
o
This was good
~','i th
.17
qvide~ee
(fi~ure ~)
t:':ds info't':'Da tion ~,noth8r eol1.,::nr. (16 x ~. b em) was:~re-
Ea.:!Jple "'as 8Dulied
th: .. t e::uilib:-'a tier:
':IBSJiS
the enzyme was
c8~in~
r~t
~ashcd
'--1).I'
"l/1t:::,·,
( d':'
_t..,...:1
. . . . 1•...;'
j
\' .
,'r"...-1~.~.I.,.~!.1
<
£",'
,.--j
,.1.' .......
tal':er:ly carrir::d 'Jut id ttl
the::rs,liieY':t ','\ras run sc)lely 1,':l th C.l
enzyme
~.
that the enzyme eluted at 0.2
:c
buffer.
BUt, a rcfr5etionstion
off at the end af the
.~radient
t'n
<).
b .,
c)u:[' fer
Eind
III h·.):.es tna t a l l
w~s
Ettemoted
a~ain an~
~ith
increasin~.
12
su
E~rn~.tar~t
oef<)t'8 diplysis
after dialysis
:Jr:)t. c:Jnc.
V81.
lC.l. G
l~ .... ~~
f~').
i._~
7 • ~t
f1:)".: r. t~;
T:'
0.Ct.
CJllr~ctillg 1,)0- ::; r:,l
ft'c..ctiJDS, cJ:~_
2.
0.33 ml/rr;in.
fracto
1-64
65
68
70
7~~
74
76
0
.75
~_. J
5.7
1 l.i_.t...~
28.
43.
•
_.4.
'18
56.
90
80
9~;
8;;;
34
53.4
56.
~j3.
96
,_I.
36
38
~~~~.• ~3
~ : '
f.., •
3
98
4.
~
d
;:)4
~._.6
le.
"
13
~;r!,:;p8.ratiun.
;:;ss&yer:i.
(t.c"b1e ;<:,)
,; • :j05 Tr i s
_~;::l
3.c
t
Z~
55~
'/,ras
vel. (4
fd.
1-2i:
() -
0 ,.
1 r.'.,
. ··
J.
·e
',·
·
.:
, ·
•
L
0
L
~;
'. b
0 L
•
:-J • /u'l'r,
.
_~.
0.1.'
1)
- s. /-_.,
0~) JJ
x .005 ,~b
dt'.d.
0
· ""
l~~'.EI
:::__ v
0
•,
·
,.
<
;
~-
r:.
'-
""
'1. .•1
.;
0
'-'
0
.:~1
.1
r'(
f'.'
t.
(:~
0
:)
LL3
50
5L
54
56
60-64.
6.3
~
...:';-
·1.5
0
.
1 • ..
:J
J.
c
t::,
v
,..;
;)
J
~.=.
0
,(..
···
',-,'
R,sS
0
,
'-
:J
t::
v'
14
J.45 ~ concentration.
haus the fact that
(fi~ure 4)
thi~
tY0S of resin shrinks ennreciahly cau56f
'ss~bility
ec:uil:: bra. ted
-----_._-----------_
..
~"'i th
to be
i~V8S-
8.05 ,- r::'ris
15
buffer.
column &..ri
Z
~l
colmer:
tr:!?
eh::ted wi th
7'8.S
O.O~)
'~
Luffer cullectiLi;
frscticns with a flow rate of approximately .5
~l/~iri.
The
assay results are as f)llows:
Fracticrl :/'
":/el.
(\
r,
"./.~. /";1"1
NJ ............
U'
x .0025 Abs./50 ul)
o
1-14
18
.8
C'.
"'")
c
~.2
c~.
5.5
e, .2
f·.5
5.3
~:.
;:'4
;:6
1. :J
. ,
~:J3-
~nce
~
.J
agair a
(fi,~ure
5)
these two
br~a~
b&r:~
is Aluted similar to the ion excharge
!~eth~d.
Purificatic,r; is 21so sir'!ilar toO tne ioOn 8xcllE,I:ge results.
t8ch7icu~s
using s slightly
time
7
:!..5
~t ~hich
the
yiel~
~ifferer:t
en?y~e
similar
r6s~lts.
graiiprt, sri
8~~lyir:~
(2ni pig~ent) elutes.
different
It is
the first rur: th-s.t it if; c::c,psclp of beiL; dis':;L3ced
"'ere rL:;t t f"r thiE toO occur.
It is
~ossible
sa~ple
'hvi~us fro~
frO!D
the column
that with each
ne~
16
7hr? errat.Lc activit;;.r seer.
lic:uiri lE:vel
i~.
all:>~';ed
t:J f·:)ll
.1. ·.'.
~l2.Y
Ultiil!: ti:::ly, tb.:; fbCt tha t it
tilis partict;lar isolati·.lr, :' te;J.
'n the wh:.le, till':
~8thld
result~;
:)f this v:ork have br:cn t;seful in
ta this oarticu13r isolation
~f
adenosine
de8~ina5e
fro~
involve roB r:y'r:')r~.': r1;ns, s !yl :NJrd puleticm of V;j rious CO;'1di tio: s.
~~uld
obtain
t;~der
sinilar conriitions, and that they are consistent
ld th 'Nha t :)thf:r researchers find at this ::mrifica ti::m s te~J.
co~paris~lD 1N:l th
.
preVl~)~.S
f~he
'Viori:
aeQ ,
':.'1 th t'(8)ne gel filtration run
points ta tjis fact.
~his
is a report uf tne
~ork d~ne
on the limited
ap:1iccti~n
17
T:~is
may a1s') be rv:;cessary t')
9~rh~_ns
th8 ':;i2:I"!:8nt could be
"·Tork in
thi~,
~.:.re8.
further study.
It
i~·
s~t
rid of the nigr.Jer:t
ch~rr:..ct~;riz8rl.
()0peri
T)rt:;~ent ,:'1'
Thsre if: l:.mcn :lore
that this 'lmrk
C2n
be used for
Table 2.
Prot. Cone.
'-1g "'rnt 7"~1
FraetLJn
....I-.?
~
~
•
.
SD. i'et.
Vel:)city
cu ....'s /"";n
l.1
U '.'
.....
,'. ri •
L.
•
.........
d. Ad.
•
uM/mg prot.
,··d.
~ot. Act.
ui,v:-:,ir.
-r::ir..
d.Ad.
17,900 -
homogenate
15.8
3.
D-401 "Oel.
596
7
1. E~~'~
5.6
1.9
C.l
~~.'~?
1,450
244
.35
!':·'}:~.5
7.1
7, :;::80
~:, ~~~O
5 .t.-'
64,000
v"
su·~.
4C)-50% ::e1.
Lj4·.3
~~6
sup.
5.2-5
'-.
,-
.
• :2
1. IJ~:.
Z~~·.l
61
1:).9
•6
1~;,
;:'00 4:,5:)0
Table
u
4.1
1:.).4
c'. (31
4.05
.31
9,15U
1,.330
;:·6.4
1::}.2
b4,~00
~;7,
5.1
2·3. ~~
4.~6
;3,000 Z.,672
6. 3;~~
17.0
~;;:.~.
llOm'Jgena te
ZO.9
,~i.
0-40~~
Del.
138
1::).1
Sllp.
'?54
6. :36
Q
40-55% ·pel.
46.8
~)f3
SL;p.
~:.6·:3
1.14
• "3
7.
~,
• 1_.:1Z~
r/
6
;), 1~~~)
r;;: ~)
,JOO
4CJO
1~,C60
Table 4
Ch r :::>:il. £'r e e t .
fr2etion
f'!J.
rct.-
/,,0.•
Chro~atogrsphy
~otal
TIurifieation
~urifie~tion
80
.05-1.0 U gradient
~'rcct Lm
45
.005-1.0
~
fr'&.ctL:m
~~8
gradient
gel fil tro.ti~)!~
406
:1.56
166
15.95
14'.)
13.47
Fii:::ure 1
eeu?.l v8lurr:es nf
-
-initi~l
buffer
- -fill'=,l buffer
- - - - - -
-m~g~etic
stirr~rs
- - - - - - - clamp
cclur:m:
tube
through stopper (edjust&ble)
-~~TIlic&tion
- - 7_ - -
liquid level
- - - -ion
exch~ng~r
DEAE- ~~ e-:,:,h;:: r1 e x
- t8 Gilson automatic
cullect,:)r
IOI~
i::.xC.dn',.GE APPil..tUiTUE,
~ith
gra1ient system
fracti~n
Figtlre
4.0
~'
3.0
2.0
Re1.
Vel.
1.0
o L -____-+__~~~----~~----~------~----_+--~--~------o
50
60
70
00
110
.·J05
.10
.12
.14
.1'1
frG.ction .1/Tris concentre,tion,
.40
~,~
Figure 3
56
48
40
- - - - - - Ad.
Re1.
Ve1.,;o:s
'-'''~
16
- - - - - (:1. Ad •
8
68
70
80
90
98
Figure 4
4
-----A.."J..
Pel.
Vel.
1
- - - d.Ad.
o ~-+~~~------~~------------+-~----------~--------~
50
60
30
40
fraction #
Figure 5
7
6
5
Pel.
Vel.
4
';':
0
"
t:::..
1
0
0
20
25
fraction #
2.:0
35
40
BIELIOCrEAPh'Y
Br2dy, ':'.:1.
"l~ ~tudy
in Six
~eaminase
of the TissDe T)istributior: of Adenosine
~ammali&n
Speci~s".
Comparative Biocheoistry
and Phi sio1ogy, vol. 14 (1965), IF). 101-12.0.
~:-isher,
J. R., VB, ,0. ? , 8r,d
Gifferer:cef in AdEr:0sir:e
Chils~m,
Dna~i~ase".
ar:.d ?hysi~)l::::gy, v()l. 16 (1965),
~a,
P.
~.,
and Fisher J.?
?hysiol:~,
',. P.
:cP.
I!PdenJsine
vol. 19 (1966),
Dr'.
l!f.ol!le
fpecific
~_'iSSU8
rO~Darative
Biocrie~istry
199-203.
~ea5ina~e--fo~e
;v21uticr:ary
799-:307.
p:;. 152'--155.
:,~.
J:iaguire, Helem
Desmins:se,
er:d J1ocb'!ell,'far,Garet.
::U!'ificati~,r:
'
ina:se
" .,,,)
' "1 ecv 1 ar
.)e8,;11
?frugner, IT.
"Etvcies on
i'iul Pro;:erties of
D'
.n~lrLJ:;CO 1 o",,~y,
,~;x
_~der"osine
2sart·:·cie:lOsine
e ~)
'~
va l• 't::>, ( 1 r,.:lUG,
Pt"
"tder::)sir:e :)eamir:asp froITi Calf rpleen ll
•
of lioche~istry and ~ioohysics, v01. 119 (1967),
'-~)'
'-"~ 7'LJ:-;:;
)'-i.
Archives
:cP.
141-154.