Excited state lifetimes of pyrimidines and purines in room temperature solution from fluorescence
quantum yields and anisotropies
by Walter Berkett Knighton
A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE
in Chemistry
Montana State University
© Copyright by Walter Berkett Knighton (1980)
Abstract:
Fluroescence excitation spectra are presented for 26 nucleic acid components in room temperature
aqueous solution. The fluorescence excitation spectra and absorption spectra coincided for all the
pyrimidines studied. Many of the purines, however, exhibited considerable deviations between the
fluorescence excitation and absorption spectra. Discussion leads to the conclusion that discrepancies
between the two spectra are not due to competition between internal conversion and vibrational
relaxation, but can be attributed to the presence of a highly fluorescent minor tautomer. Fluorescence
excitation spectra of methylated adenine derivations have contributed to the recognition of the
tautomers responsible for the fluorescence observed for adenine, in both neutral and acid solutions. The
fluorescence properties observed for adenine in neutral solution are consistent with the previously
reported 7(H) tautomer model. The protonated adenines studied indicate that the fluorescence is
probably dominated by a tautomeric species, which has protons residing on both the 7 and 9 positions.
Fluorescence lifetimes are predicted from radiative fluorescence lifetimes and quantum yield
measurements for 10 pyrimidines (8 neutrals and 2 cations) and 12 purines (8 neutral and 4 cationic
species). Reorientational times (TcSB)derived from the predicted fluroescence lifetimes (TfSB) and
fluorescence anisotropy measurements are compared to those calculated using classical Stokes-Einstein
hydrodynamic formulas (Tc stick). For the 13 nucleic acid components in which the quantum yield and
polarization were independent of wavelength the ratio of Tc SB/TC stick ranged from .1 to .7. The
average ratio was .27 for the neutral species and .5 for the cations.
Predicted rotational correlation times. (TcSB) were found to be on the order of 10 to 20 picoseconds
for the neutral free bases. Of the 13 nucleic acid components in which there was no wavelength
dependence of the quantum yield and polarization ratio 10 had predicted fluorescence lifetimes (TcSB)
which were less than 2 picoseconds. STATEMENT OF PERMISSION TO COPY
In p rese n tin g th is th e s is in p a r t ia l f u l f i l l m e n t o f the
requirem ents f o r an advanced degree a t Montana S ta te U n iv e r s ity ,
I agree th a t the L ib ra ry s h a ll make i t f r e e ly a v a ila b le f o r i n ­
s p e c tio n .
I fu r th e r agree th a t perm ission f o r exten s ive copying
o f t h is th e s is f o r s c h o la rly purposes may be granted by my major
p ro fe s s o r, o r , in his absence, by the D ir e c to r o f L ib r a r ie s .
It
is understood th a t any copying o r p u b lic a tio n o f th is th e s is f o r
fin a n c ia l gain s h a ll not be allow ed w ith o u t my w r itte n perm ission
Si gnature
Date
6 //? /^ A .
DEDICATION
I would l i k e to d e d ic a te t h is th e s is to my w if e , Maryanne,
and to my m other, f o r w ith o u t t h e i r p e r s is te n t encouragement and
a s s istan c e t h is th e s is would never have been w r it te n .
EXCITED STATE LIFETIMES OF PYRIMIDINES AND PURINES
IN ROOM TEMPERATURE SOLUTION FROM FLUORESCENCE
QUANTUM YIELDS. AND ANISOTROPIES
by
W a lte r B e rk e tt Knighton IV
A th e s is subm itted in p a r t ia l f u l f i l l m e n t
o f the requirem ents f o r the degree
of
MASTER OF SCIENCE
in
Chemis tr y
Approved:
C h airperson, Graduate Cofehiittee
f~ j.
Head, Maoor/D e p a rtment
GrSduateDeah
MONTANA STATE UNIVERSITY
Bozeman, Montana
May, 1980
iv
ACKNOWLEDGMENT
I would Tike to express my deep a p p re c ia tio n to Dr. P a tr ik R.
C a lI i s , who made t h is p r o je c t p o ssib le and who has helped b rin g my
ed u catio n al m a tu rity out o f in fa n c y .
/
I express my s in c e re thanks to Tim Aoki and Bruce Anderson
w ith whom I enjoyed the good tim es and persevered through the bad.
V
TABLE OF CONTENTS
Page
LIST OF FIGURES ......................................................................
vi
LIST OF TABLES ......................................................................... ..........................
ix
INTRODUCTION ........... .................................................. -................ : ..........................
I
EXPERIMENTAL PROCEDURE ...............
14
RESULTS ................
22
DISCUSSION .................................................................................................................
88
SUMMARY ............................
102
APPENDIX A .......................
104
LITERATURE C IT E D ......... : ...................................'......... -........................................ 106
/
•
.
/
vi
L IS T OF FIGURES
F ig u r e
Page
1.
E le c tro n ic s ta te diagram f o r a ty p ic a l molecule ........................;
2
2.
Instrum ent geometry f o r measuring p o la riz e d emission ___ . . .
4
3.
Corrected fluo rescence e x c ita tio n and absorption spectra
f o r tryptophan in n e u tra l s o lu tio n ...............................................
.4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
21
P lo t o f Ro/R - I versus
s t ic k f o r the n e u tra l nu­
c le ic a c id components in room tem perature aqueous s o l­
u tio n ........................................................................................
28-29
P lo t o f Ro/R-1 v e r s u s s t ic k f or the protonated
n u c le ic acid components in room tem perature aqueous
s o lu tio n .................................
30-31
A b sorption , flu o re s c e n c e , and co rre c te d fluo rescence ex­
c it a t io n spectra f o r adenine in pH 6 .7 aqueous s o lu tio n . .
34-35
A b sorption , - flu o re s c e n c e , and c o rrec ted fluorescence ex­
c it a t io n spectra f o r adenine in pH 1 .5 aqueous s o lu tio n . .
.35-37
A b sorption , flu o re s c e n c e , and c o rrec ted fluo rescence ex­
c i t a t i o n spectra f o r 2-m ethyl adenine in pH 1 . 5 aqueous
s o lu tio n .................................................................................
38-39
A b sorption , flu o re s c e n c e , and co rre c te d fluo rescence ex­
c it a t io n spectra f o r 3-m ethyl adenine in pH 1.5 aqueous
s o lu tio n ..............................................................
40-41
A b so rp tio n , flu o re s c e n c e , and c o rrec ted fluo rescence exc it a t io n spectra f o r N- -m ethyladenine in pH 6 .7 aqueous
s o lu tio n .....................................................
'42.-43
A b so rp tio n , flu o re s c e n c e , and co rrec ted fluo rescence ex­
c it a t io n spectra f o r N°-m ethyladenine in pH 1 .5 aqueous
s o lu tio n ..............................................
44-45
A b sorption , flu o re s c e n c e , and co rrec ted fluo rescence ex­
c it a t io n spectra fo r N °,N °-d im eth yla d en in e in pH 6 .7
aqueous s o lu tio n ..........................................
46-47
A b sorption , flu o re s c e n c e , and co rre c te d fluo rescence ex-
v ii
F ig u r e
Page
c it a t io n sp ectra f o r
N^-dim ethyladenine in pH 1.5
aqueous s o lu tio n .......................................... ................... .......................... 48-49
14.
15.
16.
17.
18.
19.
A b s o rp tio n , flu o re s c e n c e , and co rre c te d fluo rescence ex­
c it a t io n spectra f o r 7-m ethyl adenine in pH 6 .7 aqueous
s o lu tio n ........................................................................... ".............................
50-51
A b so rp tio n , flu o re s c e n c e , and co rre c te d fluo rescence ex­
c it a t io n sp ectra fo r 7-methyl adenine in pH 1 .5 aqueous
s o lu tio n ...............
52-53
A b sorption , flu o re s c e n c e , and c o rre c te d fluo rescence ex­
c it a t io n spectra fo r adenosine in pH 6 .7 aqueous s o l­
u tio n .....................
54-55
A b sorption , flu o re s c e n c e , and co rre c te d fluo rescence ex­
c it a t io n sp ectra f o r adenosine in pH 1 .0 aqueous s o l­
u tio n .................................................................................................................
56-57
A b sorption , flu o re s c e n c e , and co rre c te d fluo rescence e x ■ c it a t io n sp ectra f o r N °-m ethyladenosine in pH 6 .7
aqueous s o lu tio n ....................................................................................
58-59
A b so rp tio n , flu o re s c e n c e * and co rre c te d flu o rescen ce ex­
c i t a t i o n sp ectra f o r N6 , N °-dim ethyladenosine in pH 6 .7
aqueous s o lu tio n ........... ■.........................................................................
60-61
20.
A b s o rp tio n , flu o re s c e n c e , and c o rre c te d fluo rescence ex­
c i t a t i o n spectra f o r N °, N^-dim ethyl adenosine in pH 1.5
aqueous s o lu tio n ............................... ...................................................... ' 62-63
21.
A b sorption , flu o re s c e n c e , and c o rre c te d fluorescence ex­
c it a t io n spectra f o r 3-methyl c y to sin e in pH 4 .4 aqueous
s o lu tio n ........................................................................................................
64-65
A b sorption , flu o re s c e n c e , and co rre c te d fluorescence ex­
c it a t io n spectra f o r 5-hydroxymethyl cytosine in pH 6 .7
aqueous s o lu tio n
...............................................................................
66-67
A b so rp tio n , flu o re s c e n c e , and c o rre c te d fluo rescence ex­
c it a t io n spectra fo r 5-hydroxymethyl cytosine in pH 1.5
aqueous s o lu tio n .■......... ■.........................................................................
68-69
A b sorption , flu o re s c e n c e , and co rrec ted fluo rescence ex­
c it a t io n spectra f o r c y tid in e in pH 6 .7 aqueous s o lu tio n .
70-71
22.
23.
24.
v iii
Figure
25.
Page
A b so rp tio n , flu o re s c e n c e , and co rrec ted fluorescence ex­
c it a t io n spectra f o r c y tid in e in pH 1 .5 aqueous s o l u t io n .. .
72-73
26.
A b sorption , flu o re s c e n c e , and co rrec ted fluo rescence ex­
c i t a t i o n spectra f o r c y tid in e 5 ' -monophosphate in pH 6 .7
aqueous s o lu tio n ..........................................................................................., 74-75
27.
A b so rp tio n , flu o re s c e n c e , and c o rrec ted fluo rescence ex­
c it a t io n spectra f o r 7-methyl guanine in pH 6 .7 aqueous
s o lu tio n ......... ............................................................. ................................... ..
76-77
A b sorption , flu o re s c e n c e , and c o rrec ted fluo rescence ex­
c it a t io n spectra f o r 7 -m eth ylguanosine in pH 1.5 aqueous s o lu tio n .................... - .....................................................................................
78-79
A b sorption , flu o re s c e n c e , and c o rre c te d fluorescence, ex­
c it a t io n spectra f o r thymine in pH 6 .7 aqueous s o lu tio n . . .
80-81
A b sorption , flu o re s c e n c e , and co rre c te d fluorescence ex­
c it a t io n spectra fo r thym idine in pH 6 .7 aqueous s o lu tio n .
82-83
A b sorption , flu o re s c e n c e , and co rre c te d fluo rescence ex­
c it a t io n spectra fo r thym idine 5 1-monophosphate in pH 6 .7
aqueous s o lu tio n ..........................................................................................
84-85
Possible tautomers o f protonated ad en in e........... ................................
gg
28.
29.
30.
31.
32 .
ix
L IS T OF TABLES
Table
Page
1.
P o la r iz a tio n s , Fluorescence Y ie ld s . Fluorescence L i f e ­
tim es and R o ta tio n a l C o rre la tio n Times fo r N u cleic
Acid Components in Room Temperature Aqueous S o lu tio n .........23
2.
R atios o f ^ c ^ ^ /^ c St1' c^fo r the N u cleic Acid Components
in a M b n o to n icaIly Decreasing Order ............................................ 32
3,
Fluorescence Y ie ld s , Fluorescence E x c ita tio n Spectra
Maxima and Agreement to V a v ilo v 's Law f o r the N eutral
and Protonated Adenines in Room Temperature Aqueous
S o lu tio n ...............................................................................................
86
X
ABSTRACT
Fluroescence e x c it a t io n sp ectra are presented f o r 26 n u c le ic
acid components in room tem perature aqueous s o lu tio n . The f lu o ­
rescence e x c it a t io n s p e ctra and absorption sp ectra coincided f o r a l l
th e p yrim id in es s tu d ie d . Many o f the p u rin e s , however, e x h ib ite d
co n sid erab le d e v ia tio n s between the fluo rescence e x c ita tio n and
absorption s p e c tra .
Discussion le a d s .to the conclusion t h a t d is ­
crepancies between the two sp e ctra are not due to com petition between
in te r n a l conversion and v ib r a tio n a l r e la x a t io n , but can be a t tr ib u te d
to the presence o f a h ig h ly flu o re s c e n t minor tautom er.
Fluorescence
e x c ita tio n spectra o f m ethylated adenine d e riv a tio n s have c o n trib u te d
to th e re c o g n itio n o f the tautomers resp o n sib le f o r the fluo rescence
observed f o r adenin e, in both n e u tra l and acid s o lu tio n s .
The f lu o ­
rescence p ro p e rtie s observed f o r adenine in n e u tra l s o lu tio n are
c o n s is te n t w ith th e p re v io u s ly rep o rted 7(H ) tautomer model. The
protonated adenines s tu d ied in d ic a te th a t the fluo rescence is probably
dominated by a tau to m eric s p e c ie s , which has protons re s id in g on both
th e 7 and 9 p o s itio n s .
Fluorescence lif e t im e s are p re d ic te d from r a d ia t iv e fluo rescence
lif e t im e s and quantum y ie ld measurements f o r 10 p yrim idines (8
n e u tra ls and 2 c a tio n s ) and 12 purines (8 n e u tra l and 4 c a tio n ic
s p e c ie s ).
R e o rie n ta tio n a l tim es
d erived from th e p red icted
flu ro e scen c e lif e t im e s
) and fluo rescence an iso tro p y measurements
are compared to those c a lc u la te d using c la s s ic a l S to k e s -E in s te in
hydrodynamic form ulas ( T c 1 ;For the 13 n u c le ic ac id components
in which the quantum y ie l d and p o la r iz a tio n were independent o f wave­
len g th the r a t io o f /r,c5B/'7^s tlc K ranged from .1 to .7 . The average
r a t io was .27 f o r the n e u tra l species and .5 f o r the c a tio n s .
P red icted r o ta tio n a l c o r r e la tio n times. (T^SB) were found to be on the
order o f 10 to 20 picoseconds f o r the n e u tra l fre e bases. Of the 13
n u c le ic acid components in which th e re was no wavelength dependence o f
the quantum y ie ld and p o la r iz a tio n r a t io 10 had p red icted fluo rescence
lif e t im e s (T^sb) which were less than 2 picoseconds.
INTRODUCTION
In te r a c tio n o f m olecules, w ith r a d ia tio n in the v is ib le or
u l t r a v i o l e t region o f the spectrum, u s u a lly re s u lts in an e le c tro n ic
tra n s itio n .
Figure Ju shows a schematic o f the possib le e le c tro n ic
d e a c tiv a tio n processes a f t e r ab s o rp tio n .
s ta n ts f o r each process are also shown.
The approximate ra te con­
The n o n ra d ia tiv e processes
in clu d e v ib r a tio n a l r e la x a t io n , in te r n a l conversion and intersystem
cro ss in g .
cence.
The r a d ia t iv e processes are fluo rescence and phosphores­
V ib ra tio n a l r e la x a tio n invo lves the tr a n s fe r o f excess mol­
e c u la r v ib r a tio n a l energy to the s o lv e n t.
ra d ia tio n le s s process, whereby molecules
In te rn a l conversion is the
go from an e x c ite d s in g le t
e le c tr o n ic s ta te to th e ground s t a t e , co n vertin g e x c ita tio n energy to
h e a t.
Intersystem crossing is in te rn a l conversion in v o lv in g a change
in m u l t i p l i c i t y .
The r a d ia t iv e processes in v o lve emission o f a photon
from the low est v ib r a tio n a l le v e l o f the e x c ite d s ta te to some v ib r a ­
tio n a l s ta te o f the e le c tr o n ic ground s t a t e .
I f the emission is from
th e e x c ite d s in g le t s t a t e , i t is r e fe r r e d to as flu o re scen c e; whereas,
emission from, the low est t r i p l e t s ta te is phosphorescence.
The e x c ite d s ta te lif e t im e o f a molecule is the in verse o f the
sum o f a l l the r a te constants in volved in the e le c tr o n ic d e a c tiv a tio n
process.
is
The e x c ite d s ta te l i f e t i m e , in terms o f these r a te constants
,
. ^ f = kf+k-ic+kisc = z^
cH f
(I)
2
PHOSPHORESCENCE
lO 'M O V '
Figure I
E le c tro n ic s ta te diagram fo r a ty p ic a l molecule
3
JtfherezXrad is the r a d ia t iv e lif e t im e and ^ i s
the quantum ,e ffic ie n c y ( I )
Several research groups have rep o rted the fluorescence o f the n u c leic
ac id components to be very weak a t room tem p eratu re, having quantum
y ie ld s on the o rd e r o f 10~4 ( 2 - 4 ) .
Morgan and Daniels (5 ) have e s t i ­
mated the r a d ia t iv e l if e t im e s , using the method described by S t r ic k le r
and Berg, to be ca. 10"® f o r the n u c le ic acid components.
e x c ite d s in g le t lif e t im e s on the o rd er o f a picosecond.
This in fe rs
D aniels and
Hauswirth (2 ) have suggested th a t i f the e x c ite d s ta te lif e t im e s are
indeed th is s h o rt, then the molecule would be able to undergo l i t t l e
r e o r ie n ta tio n in the e x c ite d s ta te and should e x h ib it h ig h ly p o la riz e d
em ission.
Two authors have found the n u c le ic acid components to e x h ib it
co n sid erab le flu o rescen ce an iso tro p y in room tem perature aqueous so lu ­
tio n ( 5 , 6 ) .
I
The re s u lts o f p h o to s ele ctio n th eo ry are u t i l i z e d h e a v ily in any
sxtudy in v o lv in g fluo rescence an iso tro p y measurements.
A lb r e c h t.(7 ) has
published an e x c e lle n t comprehensive review o f p h o to s ele ctio n methods,
so a b r i e f version w i l l s u ffic e here.
Illu m in a tio n o f an is o tro p ic s o lu tio n , w ith e it h e r p o la riz e d or
u n p o larized l i g h t , leads to p h o to s e le c tio n o f an ensemble o f molecules
whose absorption d ip o les are p ro p e rly o rie n te d .
Figure 3. shows the t y ­
p ic a l in stru m en tal c o n fig u ra tio n employed f o r measuring p o la riz e d emis­
sio n .
y a x is .
E x c ita tio n is d ire c te d along the x a x is , w ith view ing along the
The p r o b a b ilit y th a t a molecule w i l l undergo p h o to s ele ctio n is
4
y
Figure 2
Instrum ent geometry f o r measuring p o la riz e d emission
5
2
given as cos 0 where 0 is the angle between the absorption d ip o le and
the d ir e c tio n o f the e l e c t r i c v e c to r o f the l i g h t .
iz a tio n measured I / I ^
The degree o f p o la r ­
is dependent on the angle subtended by the emis­
sion d ip o le , w ith resp ect to the absorption d ip o le .
I f o n ly one e le c ­
tr o n ic t r a n s itio n is in v o lv e d , the emission w i l l be from the same s ta te
as the a b s o rp tio n , w ith the th e o r e tic a l maximum o f I / I u being 2 .0 f o r
th e case when only one p o la r iz e r is used.
The th e o r e tic a l minimum is
envisaged when th e re are two absorption d ip o le s which are p erp en d icu lar
to each o th e r.
Then I yZ I^ is
.7 5 .
The l a t t e r represents a case where
absoprtion is in to one s ta te and the emission emanates from the o th e r.
The l im i t s o f the p o la r iz a tio n r a t i o 'I / I ^
when o nly one p o la r iz e r is used.
= N range from .75 to 2 .0 ,
U n fo rtu n a te ly , even
d itio n s these th e o r e tic a l l im i t s a re never observed.
under id eal con­
The m ajor causes
f o r d e v ia tio n from i d e a l i t y are energy t r a n s fe r to neighboring molecules
and r o ta tio n a l Brownian m otion.
I t would be expected t h a t , i f the p o la r ­
iz a tio n was measured using d ilu t e s o lu tio n s in a r ig id m edia, then the
above mechanisms would be in o p e r a tiv e .
Even under these co n d itio n s some
i n t r i n s i c d e p o la riz a tio n is observed w ith N = 1.81 being th e highest
p o la r iz a tio n r a t io observed in p h o to s ele ctio n studies ( 8 ) .
A lb rech t (7 )
has in s t it u t e d the use o f a random ization parameter to c o rre c t fo r these
d e v ia tio n s from i d e a l i t y .
In a n o n -rig id medium th e em itted r a d ia tio n can become d ep o la rized
by r o ta tio n a l Brownian motion o f the m olecule w h ile in the e x c ite d s ta te .
The p o la r iz a tio n measured f o r m olecules in a n o n -rig id environment is
p ro p o rtio n a l to the r a t i o o f
W h e re ^ f is the fluo rescence l i f e ­
tim e an d T c is the r o ta tio n a l c o r r e la tio n tim e associated w ith the decay
o f the flu o rescen ce p o la r iz a tio n
(9 ).
In 1933, P e rrin (1 0 ) form ulated
a r e la tio n s h ip between flu o re scen c e d e p o la riz a tio n and r o ta tio n a l Brown­
ian motion f o r the case o f a hydrodynam ically sp ecial m olecule
1/P + 1 /3 = (1/p o + 1 /3 )
( I + T zf ZTyc )
where P is the measured p o la r iz a tio n and Po is the lim it in g value in the
absence o f r o ta tio n a l m otion.
The p o la r iz a tio n P can be r e la te d to the
p o la r iz a tio n r a t io N by the expression:
Chuang and E isen th al
(9 ) have form ulated a general th e o ry f o r f lu o ­
rescence d e p o la riz a tio n due to r o ta tio n a l Brownian notion f o r an asym­
m e tric r o t o r .
I t is shown th a t the tim e dependent fluo rescence p o la r i­
z a tio n decays w ith a maximum o f f iv e exp onential components.
U n fo rtu ­
n a t e ly , u t i l i z a t i o n o f the g e n e r a lity o f t h is theory is s e v e r!y lim it e d ,
due to the d i f f i c u l t y in determ ining the magnitude and o r ie n ta tio n o f
the d iffu s io n te n s o r.
T h e ir expressions can be reduced to a more use-
a b le form , i f the n u c le ic acid components are considered to be re p re ­
sented as symmetric to p s.
This seems j u s t i f i a b l e due to th e p la n a r ity
and q u a s i-c y lin d r ic a l shape o f these m olecules.
Under these con d itio n s
o nly th re e r e la x a tio n tim es are in v o lv e d , due to the degeneracy.of the
eigenvalues o f the an g u lar momentum components d ire c te d along the sym­
7
m etry a x is .
'f
The th re e r e la x a tio n tim es are as fo llo w s :
- J_
I
- 6Dx
>
■ r f - _J_
_
'2
5DX+DZ
Y -
I
‘ 3 - 4DX+2DZ
where the Dk l S are the p r in c ip a l d iffu s io n c o e ffic ie n ts f o r r o ta tio n
about the k
a x is .
D1 = U
Where £ ^ is the f r i c t io n a l c o e f f ic ie n t
f o r r o ta tio n about the corresponding a x is .
The z ax is is assigned such
th a t i t is normal to the m oleuclar plane y ie ld in g Dx = D^ / D^.
Using
Tao's (1 1 ) equation 5 0 , the r o ta tio n a l c o r r e la tio n tim e can be expressed
as the weighted average o f the th re e r e la x a tio n times
'T^c = ( 3 /2 COs2B-'1^ rf'j + (3cos2e s in 2e)7^ + 3 /4 s in % 7 ^
(2 )
where 0 is the angle the t r a n s it io n d ip o le subtends w ith th e symmetry
a x is .
P o la rize d r e fle c ta n c e s tu d ie s , o f the n u c le ic acid components,
found t h a t the m ajor absorbance in the 260 nm range is in plane p o la r­
ized ( 1 2 ) .
T h e re fo re , 0 is 9 0 °.
The r o ta tio n a l c o r r e la tio n time is
fu r th e r s im p lifie d by s u b s titu tin g 0 = 90° in to the above eq u atio n .
f
C a llis
+ 3Z4 r3
(6 ) has determ ined the fr a c tio n a l c o e ffic ie n ts f o r the major
bases, assuming both s tic k in g and s lip p in g boundary c o n d itio n s and r e ­
ported the corresponding r o ta tio n a l r e la x a tio n tim es.
S tic k in g boundary
co n d itio n s a re known to o v e r-e s tim a te f r i c t i o n a l c o e ff ic ie n ts fo r
systems where s o lu te s iz e approaches t h a t o f the s o lv e n t ( 1 3 ) .
is l i t t l e
There
reason to b e lie v e th a t f r i c t i o n a l c o e ffic ie n ts determined
using s lip p in g boundary co n d itio n s (no in te r a c tio n between s o lu te and
s o lv e n t) are reasonable f o r hydrogen bonding molecules in aqueous ;
8
s o lu tio n .
I t is the b e l i e f o f t h is author th a t s tic k in g boundary con­
d itio n s are a b e t t e r approxim ation o f the hydrodynamic co n d itio n s u t i l ­
ize d in t h is s tu d y, and t h a t , although the magnitude o f the f r i c t io n a l
c o e ff ic ie n ts is too la r g e , the r a t io o f the c o e ffic ie n ts is rep re s e n ta ­
tiv e .
From C a l l i s 1 (6 ) r e s u lts i t is seen t h a t /Z^ = .857^ when s tic k in g
boundary co n d itio n s are assumed.
Since these two r e la x a tio n times are
sp s im ila r t h a t , w ith in experim ental e r r o r , the molecules can be assumed
to is o t r o p ic a lly r o ta te i t then appears j u s t i f i a b l e to use the P e rrin
re la tio n .
In t h is s tu d y, the P e rrin r e la tio n w il l be expressed in terms
o f the flu o rescen ce an is o tro p y
iz in g the p o la r iz a tio n P.
R, in stead o f the t r a d it io n a l form u t i l ­
In terms o f the a n is o tro p y , the P e rrin r e ­
la t io n becomes
R0 = R ( l + V ^ )
w ith Rq and R being the a n is o tro p ie s corresponding to Pq and P.
(4 )
The
a n is o tro p y R can be determ ined from the p o la r iz a tio n r a t io N from the
fo llo w in g expression
The P e rrin r e la t io n gives a measure O frC j 1Xl but no in d ic a tio n o f
the magnitude o f e it h e r q u a n tity .
e i t h e r o r w o u l d
O bviously, d ir e c t measurement o f
enable d ir e c t d eterm in atio n o f the o th e r.
As d i -
r e c t d e term in atio n o f e it h e r q u a n tity is not p o s s ib le , p r e d ic itiv e
methods must be u t i l i z e d .
R e lia b le d eterm in atio n o f h i n g e s
on the a -
b i l i t y to c a lc u la te th e p rin c ip a l f r i c t i o n a l c o e f f ic ie n t s , whereas
9
re q u ire s measurement o f th e r a d ia t iv e l i f e t im e and the quantum y i e l d .
As p re v io u s ly m entioned, th e re are p re s e n tly no hydrodynamic formulas
which adequately p r e d ic t r o ta tio n a l behavior o f small m olecules in aque­
ous s o lu tio n .
Fluorescence lif e t im e s w i l l be c a lc u la te d using the r a d i­
a t iv e lif e t im e s determ ined from S tr ic k le r -B e r g formulas and w il l be r e ­
fe r re d to as S tr ic k le r -B e r g flu o rescen ce I i f e t i m e s ^ ^ O ) .
The adequacy by which
represents the a c t u a l l y is based
c r i t i c a l l y upon two c r i t e r i a :
the e m ittin g s ta te c a r r ie s the e n tir e
o s c il la t o r s tre n g th contained w ith in th e f i r s t apparent absorption band
and th e m ajor absorbing species is the m ajor e m ittin g sp e cie s.
The v a -
i d i t y o f these assumptions has been challenged in ju s t about every paper
d e a lin g w ith th e e x c ite d s ta te lif e t im e s o f n u c le ic acid components.
The f a c t t h a t th e n u c le ic acid components are n itr o g e n - co n tain in g h e te r
ocycles which co n tain a t le a s t one p a ir o f non-bonding e le c tr o n s , leads
to the p o s s ib ilit y o f ta u to m e riz a tio n o r degenerate
t r a n s it io n s .
tt- tt*
and n-ir*
Several sets o f r e s u lts have caused con sid erable s k e p tiI
cism w ith regard to the v a l i d i t y o f these assumptions.
The g re a te s t source o f skep ticism is the s l ig h t l y red s h ifte d
co rrec ted fluroescence e x c ita tio n spectrum , w ith resp ect to the absorp­
tio n spectrum observed f o r th e m ajor bases ( 2 , 3 ) .
This is d is tre s s in g ,
as the constancy o f th e quantum y ie ld across the absorption band is so
common as to be c a lle d V a v ilo v 's law (T 4 ).
However, these bases are
found to obey Kasha's r u l e , which s ta te s t h a t only one fluo rescence
10
should be observed because the n o n ra d ia tiv e processes, le a d in g to the
low est v ib r a tio n a l le v e l o f the e x c ite d s t a t e , are much f a s t e r than the
n o n ra d ia tiv e and r a d ia t iv e processes lead in g to the ground s ta te (1 5 ) .
These d e v ia tio n s from V a v ilo v 's law may j u s t be m a n ife s ta tio n s o f the
d i f f i c u l t y in measuring quantum y ie ld s as low as 10
.
However, a t.lo w
tem peratures even g re a te r red s h if t s are observed, where quantum e f ­
f ic ie n c ie s are much h ig h e r, hence, d i f f i c u l t i e s
in experim ental measure­
ments can not be a t tr ib u t e d to the d e v ia tio n s observed ( 2 , 16, 1%).
These re s u lts have led to sp e c u la tio n o f the o r ig in o f t h is anomolous
behavior ( 6 , 12, 1 6 ).
D aniels (1 2 ) has p o s tu la te d th a t the prescence o f a h ig h ly f lu o ­
res c e n t minor tau to m eric species is resp o n sib le fo r the red s h ifte d
co rre c te d flu o rescen ce e x c ita tio n spectrum , w ith resp ect to the
absorption spectrum.
This argument appears p la u s ib le f o r the purine >
system, as th e re is co n sid erab le evidence supporting th e prescence o f
tautomers in the case o f adenine (1 7 , 1 8 ).
P f le id e r e r (1 9 ) has proposed
th a t a s im ila r tau to m eric system is also present in guanine, based on
i t s room tem perature absorption spectrum.
Although the argument has
been ap p lie d to th e p y rim id in e s , th e re are re s u lts c o n tr id ic to r y to the
prescence o f a h ig h ly flu ro e s c e n t minor tautom er.
Temperature jump
k in e t ic stu d ie s have shown th a t c y to sin e o n ly tautom erizes to the e x te n t
o f .25% ( 2 0 ) .
I f a minor tautom er is resp o n sib le f o r th e fluorescence
o f c y to s in e , i t must have a quantum y ie ld o f .01 o r g r e a te r .
This in fe r s
n
a f a i r l y long e x c ite d s ta te l i f e t i m e , hence, the fluo rescence should be
co n s id e ra b ly d e p o la riz e d .
However, several authors have reported room
tem perature p o la r iz a tio n s f o r c y to sin e and thymine to be n e a rly th a t
found in a r ig id glass a t low tem peratures ( 5 , 6 ) .
R ecently Vigney e t
a l . (2 1 ) found th a t thymine d id not v io la t e V a v ilo v 's law.
Wilson e t a l .
(1 6 ) have proposed th a t the o r ig in o f the wave­
le n g th dependence o f the quantum y ie ld s is based on a com petition between
th e in te r n a l conversion and v ib r a tio n a l r e la x a tio n d e a c tiv a tio n processes
T h e ir argument is based on the re s u lts found f o r is o la te d molecules in
the gas phase.
In low pressure gas phase experim ents, the r a te o f
in te r n a l conversion was found to in crease w ith in c re a s in g e x c ita tio n
energy above the 0 - 0
band ( 2 2 ) .
These re s u lts are exp lain ed in terms
o f the coupling between the ground and the e x c ite d Born-Oppenheimer
s ta te s w ith th e n u c le a r k in e t ic energy o p e ra to r (2 3 , 2 4 ).
Another r e s u lt which a ffe c ts the v a l i d i t y o f 7 ^ ^ , is the prescence
o f weakly allow ed t r a n s it io n s .
These weakly allowed tr a n s itio n s are o
o fte n obscured by the much more in te n s e allow ed t r a n s itio n s .
s e le c tio n r e s u lts are o fte n very h e lp fu l
Photo­
in id e n tify in g fluorsecence
emanating from a s l i g h t l y forbidden s t a t e .
The p o la r iz a tio n r a t io o f
AMP has been found to change m arkedly across the f i r s t apparent
absorption band ( 8 ) .
Although a l l o f the above discussions question the v a l i d i t y o f
r f SB|s re p re s e n ta tio n o f the a c t u a l i t
should be noted th a t
12
represents a low er l i m i t o f th e actu al fluo rescence l if e t im e .
That i s ,
th a t a l l o f the l a t t e r arguments lead to the p o s s ib ilit y o f longer l i f e ­
tim e s , but never s h o rte r.
The r a d ia t iv e l i f e t i m e , as determ ined by the
S tr ic k le r -B e r g form alism is in v e rs e ly p ro p o rtio n a l to the area under the
f i r s t absorption band.
In the case o f a tautom er or fluo rescence from
a s l i g h t l y fo rbid den t r a n s i t i o n , the e m ittin g s ta te c a r r ie s less o s c il­
l a t o r stre n g th than th a t determ ined by the in te g r a tio n o f the f i r s t
apparent absorption band.
which is too s h o rt.
This leads to a p red icted r a d ia t iv e lif e t im e
I f the v io la tio n s o f V a v ilo v 's law are due to
com petition between in te r n a l conversion and V ib ra tio n a l r e la x a tio n then
the quantum y ie ld measured f o r the 0 - 0
tr a n s itio n should be used, i n ­
stead o f th a t measured using e x c ite d l i g h t corresponding to the absorp­
tio n maximum.
V a v ilo v 's I aw,
In the absence o f mixed p o la r iz a tio n and d e v ia tio n s o f
should be an adequate re p re s e n ta tio n o f the actual
When these c o n d itio n s p re v a il the r o ta tio n a l c o r r e la tio n time
p r e d ic te d ,,T/cSB from 7 ^ ^
and th e p o la r iz a tio n r a t i o , should rep resen t
the re a l T ^ .
In t h is stu d y , the e x c ite d s ta te lif e t im e s fo r 26 n u c le ic acid
components in room tem perature aqueous s o lu tio n are ta b u la te d .
T h e ir
corresponsing r e o r ie n ta tio n a l tim es are als o compiled and subsequently
compared to r e o r ie n ta tio n a l times, c a lc u la te d from the S to k e s -E in s te in
hydrodynamic form u las.
The v a l i d i t y o f these e x c ite d s ta te lif e t im e s
are discussed in l i g h t o f the r e s u lts o f t h e i r co rrec ted fluo rescence
.
13
e x c ita tio n spe ctra and p h o to s e le c tio n s tu d ie s .
I t w i l l be shown th a t
th e p yrim idines have co rre c te d flu o re scen c e e x c ita tio n spectra which
c o in c id e w ith t h e i r absorption s p e c tra .
Many o f the p u rin e s , however,
a re shown to e x h ib it co n sid erab le d e v ia tio n between the two s p e c tra .
D iscrepancies between the co rrec ted flu ro escen ce e x c ita tio n spectra
and the absorption s p e ctra are shown to be a t tr ib u t e d to th e presence
o f a h ig h ly flu o re s c e n t minor tautom er and are not due to com petition
between in te r n a l conversion and v ib r a tio n a l r e la x a tio n .
Tautomeric
s tru c tu re s are p re d ic te d f o r adenin e, on th e basis o f c o rrec ted f lu o rescence e x c ita tio n s p e c tra , which e x p la in the flu ro e s c e n t p ro p e rtie s
observed in both n e u tra l and acid s o lu tio n s .
EXPERIMENTAL
The n u c le ic ac id components u t i l i z e d in th is study were obtained
from commercial sources as fo llo w s :
7-m ethyl adenine from Cyclo Chemical
C o rp o ratio n , 5-hydroxythym ine from P a c ific Chemical Company, 3-methyl ade­
nine from Vega Chemical C o rp o ratio n , adenosine and N®, N^-dim ethyladenine
from CaTbiochem, w ith the rem ainder being purchased from Sigma Chemical
C o rp o ratio n .
A ll compounds were used w ith o u t f u r th e r p u r if ic a t io n , as
t h e i r fluo rescence s p e c tra l band shape was v i r t u a l l y independent o f e x c it ­
a tio n w avelength, w ith the exception o f 2-m ethyl adenine and 5-hydroxythy­
mine.
In both o f the l a t t e r , s p e c tra l bandshapes remained constant when
e x c itin g from 250 to 280 nm.
However, e x c ita tio n beyond 280 nm led to a
la rg e red s h i f t in the fluo rescence .maximum.
N e ith e r compound was r e ­
p u r i f i e d , so data rep o rted w ith in corresponds to e x c ita tio n up to 280 nm.
W ater, once d i s t i l l e d , proved to be adequate as i t s background emission .
was roughly 20 times less than the prominent O-H s tre tc h Raman l in e .
A ll measurements rep o rted here were f o r aqueous s o lu tio n (ca.
4 - . I m.^ in n u c le ic acid components.
The in s tru m e n ta tio n was housed in
a constant tem perature la b o ra to ry a t 20 - 2°C , in which s o lu tio n temper­
atu res were assumed to be th a t o f the ambient room tem perature.
s o lu tio n s b u ffe re d to pH 6 .7 were .01 m olar in phosphate.
pH 1 .5 were . 15N in HgPO^.
N eutral
A c id ic so lu tio n s
15
The luminescence appartus used in. th is study was a c o lle c tio n
o f components which were arranged such th a t viewing was 90° to the pro­
pagation o f the e x c itin g l i g h t .
An Osram XBO 150 W /l high pressure
xenon lamp was employed as an e x c ita tio n source.
.This e x c ita tio n r a d i­
a tio n was then passed through a 500 mm Bausch and Lomb g ra tin g monochro­
m ator.
The fluo rescence c e ll was mounted in the c en ter o f a stage which
had movement c a p a b ilit ie s along the e x c ita tio n a x is .
The em itted l i g h t
then passed through a second monochromator id e n tic a l to the f i r s t .
De­
te c tio n was performed by an EMI9558QC p h o to m u ltip lie r tu b e , in which the
photocurrent produced was measured by a P a c ific Laboratory Model I I photo­
m eter.
Spectra were recorded on a H e w lett Packard Model 7030A x-y re c o rd -
e r.
Absorbance measurements were performed using e it h e r a Cary 14 r e ­
cording spectrom eter or a V arian Tektron s e rie s 634 spectrom eter w ith a
A25 model s t r ip c h a rt re c o rd e r.
The pH o f the s o lu tio n used were measured on a Corning model 12
pH m eter.
A fluo rescence spectrum o f the blank was recorded each time
to determ ine the e x te n t o f any flu o re s c in g im p u r itie s .
A few c ry s ta ls
o f the n u c le ic acid component were then im m ediately added to the flu o r e s ­
cence c e l l , except f o r those w ith low s o l u b i l i t i e s .
Compounds w ith low
s o l u b i li t ie s were preconcentrated in 25ml glass stoppered Erlenmeyer
16
fla s k s .
Quantum y ie ld and e x c ita tio n spectrum measurements were then
done im m ed iately, w ith o u t th e use o f the p o la r iz e r .
For the quantum
y ie ld d e te rm in a tio n , the e x c ita tio n wavelength corresponded to th a t o f
the absorption maximum.
The e x c ita tio n spectrum was recorded by s e ttin g
the view ing monochromator to the approxim ate wavelength o f the flu o r e s ­
cent maximum and vary in g the e x c ita tio n wavelength from 250 nm to the
onset o f a b s o rp tio n .
The p o la r iz a tio n r a t io was measured by using the
same e x c ita tio n wavelength as the quantum y ie ld d eterm in atio n and then
s e ttin g the view ing monochromator to the fluo rescence maximum.
With the
instrum ent in th is c o n fig u r a tio n , the x -y reco rd e r was operated in a
tim e scan mode o f .5 in /m in .
This allow ed the v e r t ic a l and h o riz o n ta l
components o f the emission to be measured by r o ta tin g the p o la r iz in g f ilm .
The background c u rre n t could also be m onitored by manually o p e ra tin g the
s h u tte r o f the e x c ita tio n monochromator.
An e n t ir e p o la riz e d fluo rescence
spectrum was also recorded f o r use in determ ining any background c o r r e c t­
io n s.
S p ectral v a r ia tio n s in the in t e n s it y o f the e x c ita tio n source
were determined by using a Rhodamine B quantum counter.
This e n ta ile d
ta k in g a 640 nm fluo rescence e x c ita tio n spectrum o f a 3 g /l s o lu tio n o f
Rhodamine B in eth ylen e glyco l
(2 5 ).
At high s o lu te c o n c e n tra tio n s , the
fluo rescence in te n s ity becomes independent o f co n cen tratio n and the s ig ­
nal becomes a fu n c tio n o f the quantum y i e ld and the lamp in t e n s it y .
The
I
17
quantum y ie ld o f Rhodamine B is constant across the absorption band,
th e r e fo r e , the fluo rescence in te n s ity is p ro p o rtio n a l to the in te n s ity
o f the e x c itin g l i g h t .
Phototube response was determ ined by p la c in g a
fr e s h ly prepared MgO screen in the fluo rescence c e l l ho ld er and measuring
the in te n s ity o f the s c a tte re d f i g h t , when both the e x c ita tio n and view ­
ing monochromators were s e t on the same wavelength (2 6 ).
C o rrectio n s due to s o lv e n t emission were not u s u a lly needed a t
th e co n cen tratio n s employed.
Minor c o rre c tio n s were made when needed,
however, the s o lv e n t emission had to be adjusted before i t could be sub­
tra c te d from the o v e ra ll spectrum.
These adjustments were made by using
the r e l a t i v e in te n s ity o f the Raman w ater l in e in both the blank and
sample s p e c tra .
I t is found th a t as the in te n s ity o f the water.Raman
l in e changes, the background changes p r o p o r tio n a lly .
S c alin g the so lven t
emission to the r e l a t i v e a tte n u a tio n o f the Raman lin e o f the b la n k , to
th a t o f the sample spectrum , y ie ld s a background spectrum th a t can be
su b tracted d i r e c t ly from th e o v e ra ll fluo rescence spectrum ( 6 ) .
The n u c le ic acid components, f o r which fluo rescence quantum y ie ld s
were q u ite low , req u ire d using s o lu tio n s in which con cen tratio n s were not
n e g lig ib le .
When s o lu tio n s o f th is nature were employed, the in te n s ity
o f the e x c itin g l i g h t was not constant through the c e l l .
C o rrection f o r
beam absorption along the e x c ita tio n ax is must be made, as a quantum count
e r only assures 'c o n s ta n t' in te n s ity a t the face o f the sample c u v e tte .
The d e te c to r does not view the e n t ir e face o f the fluo rescence c e l l , so
18
a geometry f a c t o r , depending on the e f f e c t iv e s o lid angle viewed by
the d e te c to r , must also be included ( 2 7 ) .
The t o ta l fluo rescence
sig n al detected by the p h o to m u ltip lie r tube is given as fo llo w s :
F =
where ^
_ /w (x )d I(x )
(5 )
T o
is the quantum e f f ic ie n c y , W(x) is a w eig hting f a c to r which
accounts f o r th e geometry o f the d e te c tio n system, and d l ( x ) is given
by B eer's law as fo llo w s :
'
- d l ( x ) = 2 .3 0 3 e c I0e™2 - 303ECXdx
(6 )
In te g r a tio n is c a r r ie d out form 0 to a_where a^ is the e f f e c t iv e p ath len g th o f the c e l l .
T ra n s la tio n o f the sample stage allo w s the d e te c to r to fucus
on any p o rtio n o f the c e ll along the e x c ita tio n a x is .
In t h is study,
concentrated samples were used e x c lu s iv e ly , hence, the sample stage was
adjusted such th a t th e d e te c to r viewed the f r o n t o f the c e ll b e tte r than
the c e n te r.
shape.
The p r o f il e seen, by the d e te c to r is probably gaussian in
However, under the above c o n d itio n s , the p r o f il e is adequately
represented by a t r i a n g le , w i t h . i t s apex a t the f r o n t w all o f the
c u v e tte .
I t was found th a t the geometry o f the d e te c tio n system could
be accounted f o r by the fo llo w in g w eig h tin g fa c to r :
W(x) = 1 -x /a
(7 )
S u b s titu tio n o f equations (6 ) and (7 ) in to equation (5 ) and i n t e ­
g ra tin g from 0 to a^ gives the t o t a l fluo rescence signal detected by the
19
p h o to m u ltip lie r:
F = <j>^IoG
( 8)
where
e
-2.3 03ec a
(9 )
which is the c o rre c tio n fa c t o r th a t accounts f o r the geometry o f the
d e te c tio n system and absorption p ro p e rtie s o f the s o lu tio n .
The e f f e c t iv e p athlength a^was found to be .21 cm.
This e f f e c t iv e
path len g th was determ ined by measuring the 350 nm flu o rescen ce in te n s ity
o f tryptophan a t d i f f e r e n t absorbances.
The a_value was determined by
ta k in g the r a t io o f the flu o rescen ce in t e n s it ie s o f the two tryptophan
samples and fin d in g a e f f e c t iv e p a th le n g th , which makes the r a tio s o f
t h e i r re s p e c tiv e G values equal to the r a t io o f t h e i r fluo rescence ins
t e n s it ie s .
Tryptophan has been shown by several authors to have an e x c ita tio n
spectrum which co in cid es w ith i t s absoprtion spectrum ( 4 , 1 6 ).
This
work has u t i l i z e d tryptophan as a standard to check th a t when the G
fa c t o r is employed the co rre c te d flu o rescen ce e x c ita tio n spectrum o f
tryptophan is superimposable on i t s absorption spectrum.
The co rrected
flu o rescen ce e x c ita tio n spectrum (FES) a t any given wavelength is the
product o f the absorbance and the quantum y ie ld measurement a t th a t wave­
le n g th .
The quantum y ie l d was determ ined r e la t iv e to t h a t o f Rhodamihe
B as fo llo w s :
20
_ pNAC^fRB-e RB
*f NACFr b -Gnac
00)
6RB = 1
where
and F^g are the flu o re s c e n t in t e n s it ie s a t the wavelength o f
in t e r e s t f o r the n u c le ic acid component and Rhodamine B, r e s p e c tiv e ly .
The quantum y ie ld o f Rhodamine B was taken to be I ( 2 8 ) .
Figure Z_
shows t h a t the co rrec ted flu o rescen ce e x c ita tio n spectrum o f tryp to p h an ,
using the G f a c t o r , co incides w ith i t s absorption spectrum.
Quantum y ie ld s were determ ined by comparing the r e l a t i v e areas
under the flu o re scen c e curves o f the n u c le ic acid components to th a t o f
.tryptoph an, f o r which th e quantum y ie ld was taken to be .15 (2 9 ) .
The quantum y ie l d determ ined from the fo llo w in g expression
,
= a r NAC f MAXNAC SNTNAC Gtr,yp r try p * f t r y p
fNAC
ARtry p pMAXtryp SNTtry p 6NAC 1NAC
(H )
where AR is the area under the co rrec ted fluo rescence curve a f t e r the
fluo rescence maximum has been norm alized to one, F ^
is the heig h t o f
the flu o rescen ce curve a t the flu o rescen ce maximum, SNT is the photo­
c u rre n t re q u ire d f o r f u l l
sca le d e fle c tio n and I is the r e l a t i v e
in t e n s it y o f the r a d ia tio n f i e l d , as found by the 640 nm e x c ita tio n
spectrum o f Rhodamine B.
21
Figure 3
Corrected fluo rescence spectrum ( f i l l e d
c ir c le s ) and absorption
spectrum (--------- ) o f tryptophan in pH 6 .7 aqueous s o lu tio n
RESULTS
Table I is a summary o f a l l the p e r tin e n t experim ental or derived
parameters fo r the hydrodynamic study o f the n u c le ic acid components.
A ll the values in Table I were determ ined by th is a u th o r, except fo r those
in d ic a te d by an a s te r is k or dagger.
were taken from C a llis
son and C a llis
(3 0 ).
(6 ) and a l l
The values f o r cyto sin e and guanine
the 5-m ethyl cytosine data from Ander­
The low tem perature p o la riz a tio n s re p o rted were
taken from o th e r sources, as in d ic a te d .
I t should be noted th a t low
tem perature p o la r iz a tio n r a tio s were not a v a ila b le f o r a l l the n u c le ic
a c id components s tu d ie d .
Values f o r the components, on which no ex­
perim ental work has been done, were taken to be the same as the parent
flu o ro p h o re under s im ila r c o n d itio n s .
The an iso tro p y is q u ite pronounced in a l l o f the n u c le ic acid
components, in d ic a tin g fluo rescence lif e t im e s much s h o rte r than t h e i r
r o ta tio n a l c o r r e la tio n tim es (w ith the exception o f the 5-m ethyl cyto sin e
anion and the 7 -m eth ylguanosine c a t io n ) .
In g e n e ra l, the pyrim idines
e x h ib it much hig h er p o la r iz a tio n r a tio s than the p u rin e s .
The protonated
adenines, which were su b s titu e d a t the 2 , 7 and 9 p o s itio n s , were not
included in Table I because they e x h ib ite d t o t a l l y d e p o la rize d .fluorescence
s p e c tra .
Table 3 l i s t s the quantum y ie ld s f o r these compounds, which are
seen to be s m a lle r than those o f the anion o f 5-methyl c y to sin e and the
c a tio n o f 7-m ethylguanosine.
This is in te r e s tin g since the l a t t e r exh ib ­
ite d some fluo rescence a n is o tro p y .
The ambient tem perature p o la r iz a tio n
TABLE I
P o la r i z a t io n s , fluorescence y i e l d (<|>f ) , fluorsecence l i f e t i m e s (7 ^) and r o t a tio n a l
c o r r e l a t io n time ( 7 ^) parameters f o r the nu c le ic acid components in room temperature
aqueous s o lu tio n .
To^
-r' s t ic k
’
Nucleic
Acid
Component
C
is the r a d i a t i v e l i f e t i m e ;
=7 * ° ^
is from Stokes--Enstein formula
N
No+
Ade
I . 4 6 + . Ol
3-MeAde
♦fxlO 4
T o 58ns
T f 58PS
R
Ro+
1.76
.235
.336
3.32+ .35
4 .2
1.38
3.2
36
I .56+ .0 4
1.76
.272
.336
1 . 17+.03
4 .5
.53
2.2
46
7-MeAde
I . 51+.02
1.76
.254
.336
8 .2 + .2 4
4 .8
12.1
43
6 -MeAde
1 .5 9 ± .0 4
1.76
.282
.336
.7 + .0 2
4 .0
.28
1.5
43
C-MeAde+
I . 49+.04
1.76
.246
.336
I . 2+.04
4 .9
.59
1.6
43
C-Me2Ade
I . 44+.04
1.76
.227
.336
.99+.14
4 .6
.45
.93
52
C-Me2Ade+
I . 55+.04
1.76
.268
.336
.47± .0 9
4 .7
.22
.87
52
Ado
I . 47+.08
1 .53
.239
.2 6 l(3 )
.49+.02
5.1
.25
2.7
89
C-MeAdo
I . 51+.04
1.53
.254
.261
.91+.06
5 .0
.45
15.5
99
G-Me9Ado
I . 46+.02
1.53
.235
.261
I . 51+.01
4.1
.62
5.6
109
3.9
T c 58PS T c st1ckPS
TABLE I
Nucleic
Acid
Component
N
No+
R
Ro+
(c o n tin u e d )
♦ fx lO 4
T 0SBns
TfS B p 5
T cSBps T c s t i c k PS
I .65+ .0 3
1.70
.302
.318
.76
7.7
.59
11
26
S-MeCyt+
I . 77+.02
1.79
.339
.345
.61+.02
6.2
.38
21
36
5-MeCyt*
I . 40+.01
1.75
.211
.333
5+1
10+1
5
S-MeCyt+
I . 69+.01
1.79
.315
.345
3+5
7+. 7
2.1
S-MeCyt-
1.04+.01
1.70
.026
.318
140+10
11±1
S-HmCyt
I . 47+.03
1.75
.239
.333
2 .1 5 + .0 4
13.4
S-HmCyt+
I . 70+.02
1.79
.318
.345
I . 88+.05
Cyd
I . 74+.02
1.80
.330
.350
Cyd+
I .78+ .0 3
1.80
.342
.350
CMP
I . 73+.03
1.80
.327
O
LD
CO
Gua*
I . 50+.02
1.72
.250
.324
7-MeGua
I . 47+.03
1.72
.239
.324
7-MeGuo+
I . 13+.02
1.65
.08
. 3 0 4 ( * ) 139
S-MeUra
I . 77+.02
1.81
.339
.3 5 1 ( b) I . 01+.21
fO
Cyt*
8 .7
40
22.2
40
13.8
40
2.87
7.3
41
7 .6
1.41
16.3
41
.68+06
7 .4
.51
8 .4
69
.9+.05
5 .0
.45
19.6
69
.84+.03
7 .4
.62
8 .9
94
2.8
7 .8
2.18
7.4
35.5
5 .2 8 + .6
9 .7
5.14
14.4
17.1
8.2
154
238
78
.83
23.7
48
108
36
TABLE I
R
Ro+
^ fx lO 4
CO
C
No+
CO
N
(/)
V
Nucleic
Acid
Components
(c o n tin u e d )
TfSBps
TcSSps
r Cst1ckPS
5-HmUra
1 . 71+.04
1.81
.321
.351
I . 06+.21
8.6
.91
9 .7
40
5-MeUrd
I . 78+.03
1.81
.342
.351
I . 02+.08
6 .5
.66
25.4
83
IMP
I . 78+.03
1.81
.342
.351
I . 14+.04
6 .3
.72
27.7
111
a
Wilson and C a l l i s ( 8 )
b
C a llis ( 6)
*A11 values were taken from o th er sources; cytosine and guanine, C a l l i s ( 6 ) , 5-methyl
c y to s in e , Anderson and C a l l i s ( 3 0 ) .
t A ll low temperature values were taken from references a and b. Values which were
not referenced were taken to be the same as the parent fluorophore.
26
r a t i o s o f the p y rim id in e s , in many cases, approaches the value o f the
low temperature p o l a r i z a t i o n , in d ic a t in g t h a t there is l i t t l e ,
i f any,
r e o r i e n t a t i o n in the e x c ite d s t a t e .
The quantum y i e l d s reported in Table I were determined r e l a t i v e
to trypto phan, so the u n c e r ta in ty o f these values is in the precision
o f the measurements.
The accuracy o f the values is dependent on the value
o f the quantum e f f i c i e n c y taken f o r tryptophan.
The quantum y ie ld s r e p o r t ­
ed here are compat a b le w ith those reported by o th er authors u t i l i z i n g
d i f f e r e n t standards.
The quantum y i e l d reported f o r thymine is in good
agreement w ith t h a t reported by Hauswirth and Daniels (3). and the values
f o r adenosine, c y t i d in e S'-monophosphate, and thymidine 5 ' -monophosphate
are w ith in experimental e r r o r o f those reported by Vigney and Duquesne
(4 ).
Also shown in Table I are S t r ic k le r - B e r g r a d i a t i v e l i f e t i m e s , which
were determined by in t e g r a t io n o f the s p e c tra l curves.
TheTf.^ ( t h e o r e t ­
i c a l fluorescence l i f e t i m e s ) ta b u la te d are the product o f the quantum
y i e l d and the r a d i a t i v e l i f e t i m e .
t im e s , "Cc ' s shown;
There are two r o t a t io n a l c o r r e la t io n
is found using equation 4 a n d f r o m
S to k e s -E in s te in formulas (see Appendix).
As noted, none o f these derived
q u a n t i t ie s have e r r o r l i m i t a t i o n s associated w ith t h e i r reported values.
This is not to in s in u a te t h a t the values are e x a c tly known, but r e f l e c t
the f a c t t h a t the u n c e r ta in t ie s o f these values are not d i r e c t l y measur­
27
a b le .
These derived q u a n t i t ie s have u n c e r ta in t ie s o f , probably, ten
percent (10%).
D eviations o f g r e a te r than twenty percent (20%) would
re q u ire a c o r r e l a t io n o f the e r r o r s .
What should be stressed is th a t
these r e s u lt s represent the most probable values and any conclusions
reported in t h is study are based on the most probable r e s u l t s .
Figures 4^ and 5^ show p lo ts o f 5°. _ i Vs
s tic k fo r s e le c t­
e d .n e u tr a l and c a tio n n u c le ic acid components, r e s p e c t iv e ly .
the assumption t h a t T ^ ^ B i s equal to the actual
Based on
the slope y ie ld s the
c o e f f i c i e n t f o r how much g r e a te r Y ^ s t i c k is than the actual r o t a tio n a l
c o r r e l a t io n tim e.
The s o lid l i n e in each f ig u r e represents the average
behavior o f the points p l o t t e d .
The n e u tr a ls appearing in f ig u r e 4^ have
^jfc l S which range from 10 times f a s t e r f o r CMP to 1.5 times f a s t e r f o r t h y ­
mine than t h e i r c o r r e s p o n d i n g T h e
mean behavior shown by the
s o lid l i n e in f ig u r e £ in d ic a te s ITc l S which are approxim ately fo u r times
s m alle r than the c o r r e l a t io n time p re d ic te d from s t ic k in g boundary con­
d it io n s f o r the n e u tr a ls .
The cations shown on f ig u r e 5^ vary from 4 times
f a s t e r f o r c y t id in e to 1 .4 f o r 7-methyl guanosine than t h e 'T cs t i c ^ 5 with
an average behavior o f roughly o n e -h a lf t h a t predicted by the StokesE in s te in formulas.
O v e r a l l, the cations appear to r o t a t e 2 times slower
than the n e u tr a ls .
Table 2 shows the r a t io s o f ' T sb/ ^
s tic k
n u c le ic acid components in a m onatonically decreasing o rd e r.
< c56/ ^ ? 1^
">s the inverse o f the slope o f fig u re s 4^ and Sv
fo r a ll
the
The r a t i o
As has
I
Figure 4
P in t o f Ro/R - I versus"Tf
SB
/ T^ s* 1
f or the neu tral
nucleic acid component in room temperature aqueous
s o lu tio n ;
(---------JjT ^ ave = .ETT^^tick
1.0
30
Figure 5
P lo t o f Ro/R-1 versus Y f S B / Y s t i c k
f o r the protonated
n u c le ic acid components in room temperature aqueous
s o l u t i o n ^ --------) T ^ ave = . 5 ^ s t ic k
32
TABLE 2
RATIOS O F ^ sb/ ^
s t ic k FOR THE NUCLEIC ACID COMPONENTS
IN A MONOTONICALLY DECREASING ORDER
NUCLEIC
ACID
COMPONENTS
^ c ^ ^ s tic k
NUCLEIC
ACID
COMPONENTS
<
SB
s t ic k
c
T-MeGuo+
.72
Gua
.21
5-MeUra
.66
S-HmCyt
.18
S-MeCyt+
.59
6 -MeAdo
.16
S-MeCyt+
.55
Cyd
.12
Cyt
.43
CMP
.095
S-HmCyt+
.4
Ade
.09
S -M eC yf
.34
S-MeAde+
.OS
S-MeUrd
.31
G-MegAdo
.OS
7-MeGua
.3
G-MeAde+
.04
Cyt+
.28
6 -MeAde
.035
7-MeAde
.28
Ado
.03
TMP
.25
G-MegAde
.018
S-HmUra
.24
G-MegAde+
.017
5 J vIeCyt
.22
33
been p r e d ic te d , none o f the molecules h av ez^ 5 8 l S which are longer than
t h e i r corresponding r^ s t i c k l S.
With the exception o f 7-methyl adenine,
none o f the o th er adenines were included in fig u re s 4 and 5^
These
molecules were excluded on the basis o f t h e i r apparent anomolous e x c i t ­
a tio n spectra or the r e s u lt s o f photoselection s t u d ie s .
Figures 6 ^ - 3 ^ show the absorbance spectra s o lid l i n e , corrected
fluorescence e x c it a t io n spectra f i l l e d c i r c l e s , and the corrected f lu o r e s ­
cence 'sprectra broken l i n e f o r the the n u c le ic acid components studied
by t h is author.
There is very good agreement between the absorbance and
fluorescence e x c it a t io n spectra f o r a l l
the non-adenine n u c le ic acid
components.
Table 3 shows a summary o f the experimental data f o r a l l
nine d e r iv a t iv e s s tu d ie d .
the ade­
W ithin the t a b le are given the quantum y i e l d s ,
approximate e x c i t a t i o n spectrum maximum and whether t h e i r e x c it a t io n spec­
trum coincides w ith t h e i r absorbance spectrum.
Only one o f the c a tio n s ,
N ^ , -dim ethyladenosine, showed any agreement between i t s fluorescence
e x c i t a t i o n and absorption s p e c tra .
There is a la rg e d i s p a r i t y amongst
the quantum y i e l d s o f the c a t i o n i c forms, w ith the
t iv e s and 3 -methyladenine being very low (ca. 10"^).
cations are very high (ca.
10- 2 )_
methylated d e r iv a ­
Whereas, the o th er
j n c o n tr a s t , most o f the n e u tra ls showed
good agreement between t h e i r e x c it a t io n and absorbance s p e c tra , except
adenine and N6 -m ethyladenine.
Also, the quantum y ie ld s do not show the
34
Wv
F ig u r e 6
Absorption (------- ) , fluorescence ( ---------) , and corrected
fluorescence e x c it a t io n ( f i l l e d c i r c l e s ) spectra f o r
adenine in pH 6 .7 aqueous s o lu tio n .
- 250
350 nm. 400
36
F ig u r e 7
Absorption (-------- ) , fluorescence ( ...........) , and corrected
fluorescence e x c it a t io n ( f i l l e d c i r c l e s ) spectra f o r
adenine in pH 1.5 aqueous s o lu tio n .
350 nm. 400
F ig u r e 8
Absorption (--------- ) , fluorescence ( -------- ) , and corrected
fluorescence e x c it a t io n ( f i l l e d c i r c l e s ) spectra f o r
2 - methyl adenine in pH 1.5 aqueous s o lu tio n .
"t
40
F ig u r e 9
Absorption (-------- ) , fluorescence
and corrected
fluorescence e x c it a t io n ( f i l l e d c i r c l e s ) spectra f o r
3 - methyl adenine in pH 1.5 aqueous s o lu tio n .
42
F ig u re
Absorption (—
10
) , fluorescence ( - ------ ) , and corrected
fluorescence e x c it a t io n ( f i l l e d c i r c l e s ) spectra f o r
6
N
- m ethyl a d e n in e in pH 6 .7 aqueous s o lu t io n .
250
300
— I------------------- !--------------------------k -
350 rm. 400
450
44
F ig u r e
Il
Absorption (-------- ) , fluorescence ( --------- ) , and corrected
fluorescence e x c it a t io n ( f i l l e d c i r c l e s ) spectra f o r
6
N
- m ethyl a d e n in e in pH 1 .5 aqueous s o lu t io n .
46
F ig u r e
12
Absorption (-------- ) , fluorescence ( --------- ) , and co rrected
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra fo r
6
N ,N
6
- d im e th y l a d e n in e in pH 6 .7 aqueous s o lu t io n .
48
F ig u r e
13
Absorption (-------- ) , fluorescence ( --------- ) , and corrected
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra fo r
6
N ,N
6
- d im e th y l a d e n in e in pH 1 .5 aqueous s o lu t io n .
350 nm. 400
50
F ig u re
14
Absorption (---------) , fluorescence ( --------- ) , and co rrected
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra fo r
7 - m ethyladenine in pH 6 .7 aqueous s o lu tio n .
350 nm. 400
52
Figure
15
Absorption (— — ) , fluorescence (...........) , and corrected
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra f o r
7 - methyl adenine in pH 1.5 aqueous s o lu tio n .
f
54
F ig u r e 16
Absorption (--------- ) , fluorescence ( ---------) , and co rrected
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra f o r
adenosine in pH 6 .7 aqueous s o lu tio n .
56
F ig u r e
17
Absorption 0-------- )» fluorescence ( --------- ) , and corrected
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra fo r
adenosine in pH 1 .0 aqueous s o lu tio n .
58
Figure 18
Absorption (------ —) , fluorescence ( -------- ) , and co rrec ted
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra fo r
6
N
- methyl adenosine in pH 6 .7 aqueous s o lu tio n .
sT
60
F ig u r e 19
Absorption (------- - ) , fluorescence ( -------- ) , and co rrected
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra f o r
6
N ,N
6
- dimethyl adenosine in pH 6 .7 aqueous s o lu tio n .
I
62
F ig u r e 20
Absorption (--------- ) , fluorescence ( ---------) , and corrected
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra fo r
6
6
N 1N -
d im e th y l a d e n o s in e in
pH 1 .5 aqueous s o l u t i o n .
3&) nm. 4 6 0
460
64
F ig u r e 21
Absorption (--------- ) , fluorescence ( ---------- ) , and co rrected
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra f o r
3 - methyl cytosine in pH 4 .4 aqueous s o lu tio n .
66
F ig u re 22
Absorption (-------- ) , fluorescence
and corrected
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra fo r
5 - hydroxymethyl cytosine in pH 6 .7 aqueous s o lu tio n .
68
F ig u r e 23
Absorption (--------- ) , fluorescence ( ---------) , and corrected
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) sSpectra f o r
5
hydroxymethylcytosine in pH 1.5 aqueous s o lu tio n .
F ig u re 24
Absorption (-------- ) , fluorescence ( --------- ) , and co rrected
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra f o r
c y tid in e in pH 6 .7 aqueous s o lu tio n .
72
F ig u re 25
Absorption (--------- ) , fluorescence ( ---------) , and corrected
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra fo r
c y tid in e in pH 1 .5 aqueous s o lu tio n .
F ig u re 26
Absorption (--------- ) , fluorescence ( -------- ) , and co rrec ted
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra f o r
c y tid in e 5 ' - monophosphate in pH 6 .7 aqueous s o lu tio n .
Ul
250
300
350 nm
460
-
4
—
450
76
3'
F ig u r e 27
Absorption (-----— ) , fluorescence ( --------- ) , and corrected
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra fo r
7 - methyl guanine in pH 6 .7 aqueous s o lu tio n .
250
F ig u re 28
Absorption (
- )» fluorescence ( -------- ) , and corrected
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra fo r
7 - methyTguanosine in pH 1.5 aqueous s o lu tio n . .
80
F ig u r e 29
Absorption (-------- ) , fluorescence ( --------- ) , and co rrec ted
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra f o r
thymine in pH 6 .7 aqueous s o lu tio n .
82
F ig u re 30
Absorption (------- - ) , fluorescence ( -------- ) , and corrected
fluorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra fo r
thym idine in pH 6 .7 aqueous s o lu tio n .
250
300
350 nm. 400
84
F ig u r e 31
Absorption (- -------) , fluorescence ( ---------) , and co rrected
f l uorescence e x c ita tio n ( f i l l e d c ir c le s ) spectra fo r
thym idine 5' - monophosphate in pH 6 .7 aqueous s o lu tio n .
H---------- c—
250
e ------------------- 1----------------------- 1
---------------------1—
300
350 nm. 400
450
86
Table 3
N u cliec
Acid
Component
XlO4
Coincidence o f E x c ita tio n
Spectrum w ith
Absorbance Spectrum
Approximate
E x c ita tio n
Spectrum
Maximum
Ade
3 .3 2 + .35
No
265 nm
7-MeAde
8 .2
± .24
Yes
270 nm
6-MeAde
.7
+ .02
No
270 nm
G-Me2Ade
.99 + .14
Yes
274 nm
Ado
.49 + .02
Yes
260 nm
6-MeAdo
.91 ± .06
Yes
266 nm
1.51 ± .01
Yes
275 nm
± .4
No
265 nm
-I
No
265 nm
No
275 nm
No
268 nm
± .04
No
275 nm
.47 + .09
No
280 nm
No
270 nm
Yes
270 nm
G-Me2Ado
Ade+
2 0 .7
Z-MeAde+
9 .9
S-MeAde+
1 .17 + .03
7-MeAde+
11.9
G-MeAde+
1 .2
G-Me2Ade+
Ado+
G-Me2Ado+
7 .7
.4
+
±
±
-I
.1
+ .02
SI
g re a t v a r ia tio n observed among th e c a tio n s .
Although no data appears
f o r adenines s u b s titu te d a t one p o s itio n , a cursory check was performed
on these compounds.
The catio n s o f both I -m ethyladenine and 1-methyTa-
denosine were found to be very weakly flu o re s c e n t, having quantum y ie ld s
much lower than ca. ICT^.
Borresen ( 3 1 ) , however, has rep o rted the c a tio n
o f 1-methyl adenosine to be h ig h ly flu o re s c e n t.
He was not ab le to docu­
ment the a u t h e n tic ity o f the fluo rescence o f th is compound.
This is the
o nly e x p la n a tio n fo r th is non-system atic discrepancy, as the quantum y ie ld s
rep o rted w ith in f o r the o th e r protonated adenines are c o n s is te n t w ith those
reported here.
DISCUSSION
A n alysis o f the e x c ite d s ta te behavior o f the n u c le ic acid com­
ponents r e lie s on the a b i l i t y o f S t r ic k le r and Berg's method to adequate­
l y p r e d ic t the actu al flu o rescen ce l if e t im e s .
Assuming th is method is
v a l i d , fluo rescence lif e t im e s determined using the S tr ic k le r -B e r g method
are taken to be adequate re p re s e n ta tio n s o f th e actu al fluo rescence l i f e ­
times o f the n u c le ic acid components when these molecules adhere to the
fo llo w in g c r i t e r i a :
(1 )
The quantum y ie ld is independent o f e x c ita tio n w avelength;
(2 )
The e m ittin g s ta te c a r r ie s the e n t ir e o s c il la t o r stren g th
represented by the area contained w ith in the f i r s t apparent
absorption band.
The adherence o f the n u c le ic ac id components to the above c r i t e r i a can
be assessed on the basis o f t h e i r co rre c te d fluo rescence e x c ita tio n spec­
t r a and the re s u lts o f p h o to s ele ctio n s tu d ie s .
Fluorescence e x c ita tio n s p e c tra .
The co rrec ted fluorescence ex­
c i t a t i o n spectra coincided w ith the absorption spectra o f 17 o f the 26
n u c le ic acid components s tu d ie d .
P re v io u s ly , thymine was the only n u c le ic
ac id component which had been rep o rted to obey V a v ilo v 's law ( 4 ) .
The
9 n u c le ic acid components which e x h ib ite d d e v ia tio n s between t h e i r co r­
rected fluo rescence and absorption spectra a l l were adenines.
Present
th e o rie s lead to the conclusion th a t the d e v ia tio n s from V a v ilo v 's law
89
are due to one o f th e fo llo w in g :
(1 )
The wavelength dependence o f the quantum y i e ld is due to
co m p etitio n between in te r n a l conversion and v ib r a tio n a l
r e la x a tio n ; or
(2 )
The fluo rescence observed is not from the m ajor absorbing
s p e c ie s , but due to the presence o f a h ig h ly flu o re s c e n t
minor tautom er.
Adherence o f any o f the n u c le ic acid components to V a v ilo v 's law
d is c r e d its th e p o s s ib il it y th a t the d iscrepancies observed can be a t t r i b ­
uted to co m p etitio n between in te rn a l conversion and v ib r a tio n a l r e la x a ­
t io n .
Other evidence a g a in s t t h is mechanism is the r e s u lt o f the c a tio n
o f 7-m ethyl ad enin e, in which the c o rre c te d fluo rescence e x c ita tio n spec­
trum is blue s h if t e d , r e l a t i v e to the absorption spectrum.
This would
in d ic a te th a t the quantum y ie ld increases w ith in c re a s in g e x c ita tio n
energy above the 0 - 0
band, which is d ir e c t c o n tra d ic tio n to th is theory
The above evidence in d ic a te s th a t d e v ia tio n s from V a v ilo v 's law , by the
n u c le ic acid components in room tem perature aqueous s o lu tio n , are not
due to com petition between n o n ra d ia tiv e d e a c tiv a tio n processes.
D e via tio n s between the co rre c te d fluo rescence e x c ita tio n and ab­
so rp tio n sp ectra are a t tr ib u t e d to the presence o f a h ig h ly flu o re s c e n t
minor tautom er.
This e x p la n a tio n has been c ite d many tim es to e x p lain
the v io la tio n o f V a v ilo v 's law and sometimes in d is c r im in a te ly ( 2 , 3 , 12,
17, 3 1 ).
Caution must be used in invoking t h is argument, th a t the d is ­
90
crepancies observed are re a l and not due to improper in te r p r e ta t io n o f
th e d a ta .
s tr in g e n t.
The tautom er model appears very g e n e ra l;
however, i t is ra th e r
I f the flu o rescen ce is due to a minor tautom er, i t must ad­
here to the fo llo w in g c r i t e r i a :
(1 )
The minor tautom er must have pK values s im ila r to the main
tautom er; and
(2 )
The minor must be present in s u b s ta n tia l q u a n tity because
o f the extreme tem perature dependence o f the quantum
y ie ld
(3 )
( 1 6 ) ; and
The p o la r iz a tio n measured should be somewhat d e p o la riz e d ,
owing to the lo n g er liv e d e x c ite d s ta te o f the minor ta u ­
tomer.
The purines stu d ied h e re , which v io la t e V a v ilo v 's law , are c o n s is te n t
w ith t h is m odel.
„
Protonated adenin e, 7-m ethyl adenine and adenosine e x ­
h ib ite d t o t a l l y d e p o la rize d flu o rescen ce s p e c tra .
The apparent quantum
y ie ld s f o r these compounds are not la rg e enough to account f o r th is de­
gree o f d e p o la riz a tio n .
The rem aining purines e x h ib ite d p o la r iz a tio n
r a tio s which a re lower than would be p re d ic te d on the basis o f t h e ir ap­
paren t quantum y ie ld s and r a d ia t iv e l if e t im e s .
,None o f th e pyrim idines
stu d ied here showed any d e v ia tio n s from V a v ilo v 's law .
This generates
sp e cu latio n concerning the v a l i d i t y o f D aniels and Hauswirth (2 ) r e s u lt
th a t the fluo rescence observed f o r cyto sin e was due to the presence o f
a h ig h ly flu o re s c e n t minor tautom er.
K in e tic studies show th a t cytosine
91
o n ly tautom erizes to an e x te n t o f .25% in room tem perature aqueous so­
lu t io n .
I f the quantum y ie ld rep o rted f o r cytosine is o nly apparent,
then the quantum y ie l d re p o rted o f the tautom er must be g re a te r than
.0 1 .
In the absence o f some phenomenally long r e o r ie n ta tio n a l tim e , the
fluo rescence should be s ig n i f i c a n t l y d e p o la riz e d .
C a llis
( 6 ) , however,
has shown c y to sin e e x h ib ite d h ig h ly p o la riz e d flu o re s c e n c e .
On t h is
eviden ce, and th e behavior observed f o r the o th er p y rim id in e s , cyto sin e
would be p red icted to obey V a v ilo v 's law .
.
Discussion here leads to the
conclusion t h a t .t h e n u c le ic acid components u s u a lly adhere to V a v ilo v 's
law and d e v ia tio n s from t h is beh avior can be a t tr ib u te d to the presence
o f a h ig h ly flu o re s c e n t minor tautom er.
F luorescent lif e t im e s p re d ic te d from the S tr ic k le r -B e r g formulas
f o r the p u rin e s , which have d e v ia tio n s between t h e i r co rre c te d f lu o r e s ­
cence e x c ita tio n and absorption s p e c tra , are not re p re s e n ta tiv e o f the
actu al flu o rescen ce lif e t im e s .
T h e ^ f ^ 's
are much too s h o rt, as t h e i r behavior is
p re v io u s ly mentioned.
reported f o r these molecules
in c o n s is te n t w ith the c r i t e r i a
.
The quantum y ie ld s not only changed across the ab­
so rp tio n band, but the values rep o rted re p res en t the apparent quantum
y ie ld .
The actu al quantum y ie ld s are much h ig h e r.
This is obvious from
the f a c t th a t the minor tautom er only absorbs a small percentage o f the
e x c itin g l i g h t , y e t is resp o n sib le f o r n e a rly a l l o f th e em ission. . The
r a d ia t iv e lif e t im e s are not r e p r e s e n ta tiv e , because the o s c il la t o r s tre n g th
is c a lc u la te d from the f i r s t apparent absorption band, which is the sum
92
a f the absorbances o f a l l the tau to m eric species.
The e x c ite d s ta te l i f e ­
tim es rep o rted f o r adenin e, N^-m ethyladenine, the cations o f 3-methyl ade­
nine., N^-methyTadenine, and N® ,N^-dim ethyladenine are anomolously sh o rt
and have no physical s ig n ific a n c e .
P h o to s e le c tio n .
The v a l i d i t y o f the p red icted fluo rescence l i f e ­
tim es r e l ie s h e a v ily on the re s u lts o f p h o to s e le c tio n s tu d ie s .
The
S tr ic k le r -B e r g method o f determ in ing the r a d ia t iv e lif e t im e s only remains
v a lid i f the f i r s t apparent absorption band contains one t r a n s itio n d ip o le .
Under these co n d itio n s m olecules e x h ib it p o la r iz a tio n r a tio s which are
independent o f e x c ita tio n w avelength.
Mixed p o la r iz a tio n
(wavelength
dependent p o l a r i z a t i o n ) , however, in d ic a te s absorption in to two s ta te s ,
where the low est energy s ta te is resp o n sib le fo r the em ission.
R a d ia tiv e
lif e t im e s determ ined f o r m olecules which e x h ib it mixed p o la r iz a tio n w il l
be too s h o rt, as th e t o t a j o s c il la t o r s tre n g th is represented by the area
contained w ith in the low est energy t r a n s it io n .
Wilson and C a llis
shown th a t AMP e x h ib its co n sid erab le mixed p o la r iz a tio n .
(8 ) have
This mixed p o la r ­
iz a tio n has been a t tr ib u t e d to the presence o f a weakly allow ed t r a n s it io n .
This t r a n s it io n is low er in energy than the more in ten se allow ed tra n s ­
i t i o n , and is resp o n sib le f o r the flu o re s c e n c e .
Adenine, more p ro p e rly
i t s flu o re s c e n t tautom er, does not e x h ib it any mixed p o la r iz a tio n , so sub­
s t it u t io n o f the 9 p o s itio n o f adenine leads to fluo rescence from a weakly
C.
allow ed t r a n s it io n .
The actu al lif e t im e s f o r adenosine, N -m ethyladeno­
s in e , IN®,N^-dimethyladenosine and N®,N®-dimethyladenine are con sid erably
la r g e r than those rep o rted here.
N^5N^-dim ethyladenine is included be­
cause i t is b e lie v e d to have a hydrogen re s id in g on the 9 n itro g e n .
Hydrodynamic b e h a v io r.
The rem aining n u c le ic acid components,
a l l the p y rim id in e s , and the 3 seven s u b s titu te d purines e x h ib ite d be­
h a v io r c o n s is te n t w ith the c r i t e r i a e s ta b lis h e d fro m .th e S tr ic k le r-B e r g
form alism .
The S tr ic k le r -B e r g fluo rescence lif e t im e s rep o rted fo r these
molecules are considered to be good re p re s e n ta tio n s o f the actu al f lu o r e s ­
cence lif e t im e s .
Whentjr58 is equal to the a c t u a l t h e n
the r o ta tio n a l
c o r r e la tio n tim e c a lc u la te d from the flu o re s c e n t lif e t im e and fluorescence
an iso tro p y represents the actu al r o ta tio n a l c o r r e la tio n tim e .
Due to the
s tr u c tu r a l s i m il a r it y o f the n u c le ic acid components, these molecules
would be expected to e x h ib it s im ila r hydrodynamic beh avio r.
o f the
re p re s e n ta tio n o f the actu al
The v a l i d i t y
can be checked by the re a ­
s o n a b ility o f the r e o r ie n ta tio n a l tim es c a lc u la te d from the P e rrin r e l a ­
t io n .
TheY cSB, g rep o rted in Table I ,
fo r these m olecules, are roughly
e q u iv a le n t, showing d e v ia tio n s o f only about a fa c to r o f th r e e .
These
d e v ia tio n s may be due to minor s tr u c tu r a l d iffe r e n c e , and p o s sib ly d i f ­
fe r e n t degrees o f s o lv e n t in te r a c tio n between the n e u tra l and protonated
sp ecies.
the Y c
These s tr u c tu r a l v a r ia tio n s can be accounted fo r by comparing
' s .to the r o ta tio n a l c o r r e la tio n times determined from the Stokes-
E in s te in hydrodynamic fo rm u las.
The S to k e s -E in s te in form ulas account
f o r the d iffe re n c e s in the volume and shape o f the m olecules.
£ and _5 show p lo ts o f
-jp —
I versus
s t ic k .
Figures
The P e rrin r e -
94
la t io n shows th a t
is equal to the actu al r a t io O f Y f A f .
fig u re s a r e , th e r e fo r e , a c tu a lly graphs O f Y f A f
c
These
versus. Y SB /Y s t lc k .
f
c
S inceY f SB i s taken to be the actual Y f , then the slope o f the lin e
y ie ld s how much la r g e r
o f th e r a t io s o f Y
c
Ycst ic k
^b/ Y
c
-js th an Yc
- Table
s t ic k f o r an
^ c 5B1s f o r the m olecules, in which
range from
2is
a com pilation
the molecules s tu d ie d .
The
Y f SB is taken to be the a c tu a l Y f ,
. l Y cstl' CK to • / Y cs^lc* .
The n u c le ic acid components fo r
which the Y f SB1S were a l l shown to be too s h o rt, y ie ld Y c^ 1S which are
not reas o n ab le, being up to 5Q tim es f a s t e r than Y ^ t i c k ^
y^^le 2 shows
t h a t , on the average, the catio n s e x h ib it longer r e o r ie n ta tio n a l times
than the n e u tr a ls .
The c a tio n s , being a charged sp e c ie s , probably ex­
perience more s o lv e n t in te r a c tio n . The hydrodynamic behavior rep o rted .
f o r these molecules appears reasonable and is in e x c e lle n t
agreement
to the hydrodynamic behavior rep o rted by Bauer e t a l . (1 3 ) f o r the c a r­
b o x y lic acids in w a te r.
The evidence p ro vid ed , e ith e r actual o r conse­
q u e n tia l, lends v a l i d i t y to the adequate re p re s e n ta tio n o f Y f BB fo r the
a c tu a lY f •
That i s , th a t some o f the n u c le ic acid components may, i n ­
deed, have fluo rescence lif e t im e s as s h o rt as one picosecond.
S ig n ific a n c e o f fluo rescence l i f e t i m e s .
Existence
o f flu o r e s ­
cence l i f e t im e s , on the o rd er o f one picosecond, is o f in t e r e s t from a
chemical physical s ta n d p o in t, as lif e t im e s th is sh o rt confirm the presence
o f exceedingly f a s t n o n ra d ia tiv e processes.
A l if e t im e o f 10
-12
seconds
in fe r s a r a te o f a t le a s t 1 0 ^ s " ^ f o r the r a te o f in te rn a l conversion.
95
_As t h is work found V a v ilo v 's law to be obeyed in the absence o f ta u tomer s , the r a te o f v ib r a tio n a l r e la x a tio n must be g t le a s t 10
v ib r a tio n a l r e la x a tio n ( 3 2 ) .
s- "1 f o r
The r e s u lts o f fluo rescence lif e t im e s th is
s h o rt are o f co n sid erab le in t e r e s t to p h o to b io lo g is ts .
I f the behavior
o f DNA is taken to be s im ila r to th a t o f the in d iv id u a l n u c le ic acid
components, the p o s s ib ilit y o f s in g le t energy tr a n s fe r in DNA is improb­
ab le ( 2 ) .
Under the most fa v o ra b le c o n d itio n s , the energy tra n s fe r r a te
is estim ated to be ca. IO+12S-1
(3 3 ).
F luorescent tautomers o f ad en in e.
Study o f the n u c le ic acid
components is undertaken in attem pt to b e tte r understand the behavior
o f DNA i t s e l f .
Much o f th is study has aluded
minor tau to m eric species in the adenines.
know what tau to m eric species are
p e r tie s observed.
to a h ig h ly flu o re s c e n t
I t is o f g re a t in t e r e s t to
resp o n sib le f o r the fluo rescence pro­
These tautomers have much longer flu o re s c e n t l i f e ­
tim es than the m ajor n o n flu o rescen t component.
So, i f the s tru c tu re o f
these tautomers is com patible w ith the s tr u c tu r a l p o s s ib ilit ie s o f the
adenine m oiety in DNA, the consequences could be profound.
These longer
liv e d e x c ite d species could c o n trib u te g r e a tly to photochemical re a c tio n s .
T h e re fo re , knowledge o f the s tru c tu re o f the fluo rescence tautom er is es­
s e n t ia l.
Spectroscopic stu d ies o f t e n ^u t i l i z e m ethylated d e r iv a tiv e s to
c h a ra c te riz e systems.
S u b s titu tio n o f a hydrogen by a methyl group, ty p ­
i c a l l y , has less than an o rd er o f magnitude e f f e c t on r a d ia t iv e and
r a d ia t iv e p ro p e rtie s o f p la n a r conjugated molecules (1 4 , 3 4 ).
non-
G en erally
96
speaking, one observes an approxim ate s h i f t o f 5 nanometers in absorp­
tio n and fluo rescence maxima, w ith the s u b s titu tio n o f a hydrogen by a
m e th y l.
Table 3 shows th a t adenine and N®-methyladenine are the only
n e u tra l adenines which e x h ib it d e v ia tio n s between t h e i r co rrec ted flu o r e s ­
cence e x c ita tio n and absorption s p e c tra .
Previous discussion concludes
these d e v ia tio n s are due to the presence o f a minor flu o re s c e n t tautom er.
Temperature jump k in e t ic stu d ies in d ic a te th a t in n e u tra l aqueous s o lu ­
tio n the 9(H ) and 7(H) tautomers are the only tautom eric species present
in s ig n if ic a n t q u a n titie s
(1 8 ).
The flu o re s c e n t p ro p e rtie s observed f o r
adenine and N^-m ethyladenine should be compared to adenosine and 7-m ethy1 adenine, which are the s u b s titu te d d e r iv a tiv e s which re p res en t the 9(H)
and 7(H) tautom ers, r e s p e c tiv e ly .
The apparent quantum y ie ld s o f ad en in e,
and .N^-methyladenine are both g re a te r than the quantum y ie ld s fo r adeno­
s in e .
The flu o rescen ce e x c ita tio n spectra o f adenine and N^-methyladenine
co in cid e w ell w ith the fluo rescence e x c ita tio n spectrum o f 7-m ethyladen in e , in both shape and p o s itio n .
The 5 nm s h i f t between the fluo rescence
e x c ita tio n maxima f o r adenine and 7-m ethyl adenine is due to the s u b s titu ­
tio n o f a hydrogen by a methyl group.
The re s u lts conclude th a t the 7(H)
tautom er is resp o n sib le f o r the flu o re s c e n t p ro p e rtie s observed fo r ade-
6
nine and N -m e thylad enine.
This conclusion is in harmony w ith o th er s tu d ­
ie s which proposed the 7(H ) tautom er is the flu o re s c e n t sp e c ie s , as the
9(H ) tautom er is r e l a t i v e l y non flu o rescen t (1 2 , 17, 3 5 ).
D im eth ylatio n
o f the e x tra n u c le a r n itro g e n does not show the presence o f any tautom er-
97
iz a t io n .
The fluo rescence spectrum is seen to be very s im ila r to i t s
s u b s titu te d analog N ,N -d im eth ylad en o sin e.
9
A p p a re n tly , the presence
o f th e two methyls on the e x tra n u c le a r n itro g e n e f f e c t iv e ly block sub­
s t it u t io n o f the 7 p o s itio n .
This, e x p lain s the s i m il a r it y between
N ^,N^-dim ethyladenine and N®,N®-dim ethyladenosine.
C h a ra c te riz a tio n o f the c a tio n ic adenine system is con sid erably
more d i f f i c u l t than the n e u tr a ls .
P ro to n a tio n leads to many more ta u to ­
m eric p o s s i b i l i t i e s , since the adenine m oiety can have a hydrogen on e it h e r
the 7 o r 9 p o s itio n before p ro to n a tio n , and then can protonate on any o f
the o th e r fo u r rem aining n itro g e n s .
Borresen (31) studied th is system
some tim e ago and concluded th a t flu o re s c e n t species had th re e hydrogens
on the exo cyclic n itro g e n and one on the 7 p o s itio n .
U n fo rtu n a te ly ,
Borresen's argument is based only on the re s u lts o f adenine and 7-m ethylad en in e and does not account fo r the re s u lts observed f o r adenosine.
Giskaas (3 6 ) has determ ined the flu o re s c e n t lif e t im e o f p ro to nated adenosine, a t room tem p eratu re, from Stern-Volm er quenching exp er­
iments to be lx l0 " 9 s .
The fluo rescence e x c ita tio n s sp ectra o f protonated
adenosine does not co in cid e w ith i t s absorption spectrum, so th is f lu o r e s ­
cence lif e t im e corresponds to the tautom er respo nsible f o r the fluo rescence
observed.
Assuming a r a d ia t iv e lif e t im e o f ca. SxlCH^s,, y ie ld s a quantum
y ie ld o f ca.
.2 f o r th is tautom er.
This in d ic a te s th a t th is tautomer
needs to be present in only small amounts to account f o r the apparent
quantum y ie ld s rep o rted f o r the protonated adenines.
/
Figure 32^ shows a l l
the p o ssib le tautom er species o f p ro to -
98
P ossible tautomers o f protonated adenine
99
Figure 32 (co n tin u ed )
100
nated adenine.
I t is n o t r e a d ily apparent which tautom eric s tru c tu re s
are represented by the various m ethylated d e r iv a tiv e s .
Al I the m ethyl­
ated d e r iv a tiv e s assume two or more o f the s tru c tu re s shown.
Since th is
is not an exh austive s tu d y , some o f the tautomers do not have c o rre ­
sponding s u b s titu te d d e r iv a tiv e s .
Because o f t h i s , i t is more useful
to e lim in a te those s tru c tu re s which can not be resp o n sib le f o r the
flu o re s c e n t p ro p e rtie s observed.
Both I -m ethyladenine and I -m e th y l-
adenosine were, found to be nonfluorescent. having quantum y ie ld s less
than 5x10"^.
I -M ethyladenosine is represented by the s tru c tu re la b e le d
( 1 ,9 ) so t h is s tru c tu re can be e lim in a te d as a p o ssib le flu o re s c e n t
tautom er.
I-M e th y la d e n in e has th e p o s s ib ilit y o f e x is tin g as the
s tru c tu re la b e le d ( 1 , 3 ) ,
( 1 ,7 ) and ( 1 ,6 ) so these, s tru c tu re s can be
excluded as flu o re s c e n t tautom ers.
The e x o cyclic n itro g e n can also
e x is t in the imino form , so the I -m ethyladenines could also e x is t as
the s tru c tu re s la b e le d ( 1 , 3 , 7 ) ,
( 1 , 3 , 9 ) and ( 1 , 7 , 9 ) .
3-M ethyl adenine
is somewhat more flu o re s c e n t than the corresponding one s u b s titu te d
adenine.
The flu o re scen c e y ie ld is s t i l l
f a i r l y low so th a t s u b s ti­
tu tio n o f the 3 p o s itio n leads to a le s s flu o re s c e n t species.
e lim in a te s the tautomers la b e le d ( 3 ,7 ,) , ( 3 , 9 ) ,
This
( 3 ,6 ) and ( 3 , 7 , 9 ) .
The o nly s tru c tu re s which remain a re s u b s titu te d a t both the 7 and 9
p o s itio n s , or in volved the exo cyclic n itro g e n .
S u b s titu tio n o f the
exo cyclic n itro g e n d e a c tiv a te d the flu o re s c e n c e , which in d ic a te s th is
n itro g e n may be in vo lved in the flu o re s c e n t tautom er,
I
D im eth ylatio n o f
101
the e x o c y c lic n itro g e n fix e s the n itro g e n in the amino form .
fluo rescence y ie ld s measured f o r these compounds appear
The low
to in d ic a te
th a t the e x o c y c lic n itro g e n may assume the imino form in the f lu o ­
resc en t tautom er.
I f the e x o c y c lic n itro g e n is in the imino, form ,
then e it h e r the I o r 3 p o s itio n must be s u b s titu te d .
However, both
I -methyl adenine and 3-m ethyl adenine had very low quantum, y ie ld s .
Methyl
. a tio n o f t h is e x o cyclic n itro g e n increases the b a s ic ity o f th is group.
I t may be th a t t h is in crease in b a s ic it y causes th is n itro g e n to become
p r e f e r e n t ia lly protonated over any o f the o th e r s it e s .
This in d ic a te s
th a t the e x tra n u c le a r n itro g e n is not in vo lved in the flu o re s c e n t ta u to ­
mer.
I t appears th a t i f o nly one tautom er is resp o n sib le f o r the f lu o ­
rescence p ro p e rtie s o f protonated ad enin e, i t must be s u b s titu te d a t
both the 7 and 9 p o s itio n to be c o n s is te n t w ith the re s u lts o f the
7-m ethyladenine and adenosine.
F u rth e r j u s t i f i c a t i o n f o r t h is s tru c tu re
is borne out by th e r e s u lts rep o rted f o r protonated 7-m e th y lguanosine,
which is the guanine analog o f the tautom er.
As shown in Table I , the
c a tio n o f 7-m ethylguanosine has a f a i r l y high quantum e f f ic ie n c y ,
although not as high as t h a t p re d ic te d f o r t h is tautom er.
Although
most o f the evidence is c o n s e q u e n tia l, i t is the b e l i e f o f t h is author
th a t the flu o re s c e n t tautom er resp o n sib le f o r the fluo rescence
p ro p e rtie s observed f o r protonated adenine has a proton r e s id in g on
both the 7 and 9 p o s itio n .
SUMMARY
The co rrec ted fluo rescence e x c ita tio n spectra coincided w ith the
absorption sp ectra f o r 10 pyrim id in es and 7 purines o f the 26 pyrim idines
and purines s tu d ie d .
The 9 n u c le ic ac id components which e x h ib ite d de­
v ia tio n s between t h e i r co rre c te d flu o rescen ce e x c ita tio n and absorption
spectra were a l l adenines.
These d is c re p a n c ie s , between the two spectra
observed f o r the adenines, were a t tr ib u t e d to the presence o f a h ig h ly
flu o re s c e n t minor tautom er.
Fluorescence e x c ita tio n spectra o f the methy­
la te d adenine d e r iv a tiv e s were u t i l i z e d to id e n t if y the flu o re s c e n t ta u ­
tomers o f ad enin e, in both n e u tra l and ac id s o lu tio n s .
The flu o re s c e n t
p ro p e rtie s observed f o r adenine in n e u tra l s o lu tio n were c o n s is te n t w ith
the p re v io u s ly proposed 7(H ) tautom er model.
The protonated adenines
studied in d ic a te d the fluo rescence was probably dominated by a tautom er
which has protons re s id in g on both the 7 and 9 p o s itio n s .
The combined re s u lts o f the co rre c te d fluo rescence e x c ita tio n
spectra and previous p h o to s e le c tio n s tu d ie s , fo r the f i r s t tim e , gave
some physical basis to the v a l i d i t y o f fluo rescence lif e t im e s p re d icted
from the i n t r i n s i c fluo rescence lif e t im e s and quantum y ie ld s f o r the
n u c le ic acid components.
The p re d ic te d fluo rescence lif e t im e s o f the
purines and pyrim id in es were found to be very s h o rt, on the order o f
IO- H
_ IO- IZ g , except f o r the anion o f 5-m ethyl cytosine and the c a tio n
o f 7-m ethyl guanosin e .
These p re d ic te d fluo rescence lif e t im e s were taken
103
to be good re p re s e n ta tio n s o f the actu al fluo rescence lif e t im e s o f a l l
the p yrim idines and the 3 purines s u b stitu ed a t the 7 p o s itio n .
The
p re d ic te d lif e t im e s o f these m olecules, in conjunction w ith flu o re s c e n t
an iso tro p y measurements,
y ie ld e d r e o r ie n ta tio n a l times c o n s is te n t
w ith the re s u lts o f o th e r small molecules in aqueous s o lu tio n .
The
n e u tra l p yrim id in es and purines appeared, on th e .a v e ra g e , to r o ta te fo u r
tim es f a s t e r than p re d ic te d from the S to k e s -E in s te in model.
The c a tio n ic
species appeared to experience more s o lv e n t in te r a c tio n , having rates
o n ly tw ice as f a s t .
APPENDIX A
D eterm ination o f ^ cS t ic k .
As p re v io u s ly shown, the r o ta tio n a l c o r­
r e la tio n tim e fo r an o b la te e l li p s o i d , f o r which the absorption is in
plane p o la r iz e d , is expressed as fo llo w s :
<
W h e r e zT 1 =
1/ 6 D „ ; zT 0 =
1
3
1
_
- Vt1 + 3 /4 r-3
;
D
W zDi :
= k T
rV
where D^ is the p r in c ip a l d iffu s io n c o e f f ic ie n t f o r r o ta tio n about the
kth ax is and ^
is the f r i c t i o n a l c o e f f ic ie n t fo r r o ta tio n about the
corresponding a x is .
-
The f r i c t io n a l , c o e f f ic ie n t f o r a hydrodynam ically s p h erica l mole­
cu le is 'g iv e n as fo llo w s :
^ o s tic k = GrV
where 1-1 is the v is c o s it y , was found to be 1 .0 centapoise a t 293° K ( 3 7 ) ,
and V is the m olecular volume which was determ ined using Van der Waals
increm ents ( 3 g ) . P e r r in 's (3 g , 40). form ulas .enable I
s t ic k to be found by
m u ltip ly in g | 0s t ic k by a fa c to r which r e la te s an o b la te e llip s o id to a
sphere o f equal volume, and depends on the a x ia l r a tio s o f the e llip s o id
(4 1 ).
The a x ia l r a t io in the xy plane was determined using the geometric
mean o f an e llip s o id and the thickness in the z d ir e c tio n was taken as
3 .4 0A.
The geom etric mean was determ ined as fo llo w s :
105
V = 4 /3 irabc (volume o f an e llip s o id )
bc =
4 ira
be = geom etric mean
V s t ic k
were determ ined using the a x ia l r a t io
k
defined as
a
bc
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