- Email: [email protected]
Fluorescence and protein structure
()
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 35005 FLUORESCENCE AND P R O T E I N STRUCTURE X. R E A P P R A I S A L OF SOLVENT AND STRUCTURAL EFFECTS ROBERT \¥. COX,VGILL The Bowman Gray School of Medicine, Wake Forest College, Winston-Salen~, N. C. ( U.S.A .)
(Received June 3rd, 1966)
SUMMARY I. Quantum efficiencies of fluorescence and emission maxima were measured in a number of solvents for tryptophanyl and tyrosyl peptides, as well as simpler derivatives of indole and phenol. Fluorescence was internally quenched by carbonyl groups but this occurred only when the molecule was solvated by a hydrogen-bonding solvent such as water or an alcohol. In non-polar organic solvents, quenching by the carbonyl group was lost and fluorescence rose to values of the parent indole and phenol compound. 2. Fluorescence quenching occurred with derivatives that contained unionized acid, amide, ester and peptide groups. These groups, when present on other solute molecules, did not quench; that is, external quenching of indole and phenol by amides, esters, etc., did not occur. An amino group alone had no effect on fluorescence, but in the free amino acids and peptides an c~-amino group enhanced the quenching action of a neighboring carbonyl group. Fluorometric titrations showed that this enhancement of the carbonyl quenching was in the order ~-NHa + > ~-NH 2 > H. 3. The bearing that these observations may have on fluorescence of proteins was discussed. A preliminary classification scheme for exposed and buried tryptophanyl and tyrosyl residues was described and a range of values was selected for the fluorescence efficiency of each type of residue.
INTRODUCTION One of the aims of this series of papers has been to define the conditions that affect the fluorescence output of tryptophanyl and tyrosyl residues so that predictions may be made of the chemical bonding and physical environment of these residues within or on the surface of a protein. The first two papers1, 2 demonstrated that the incorporation of these amino acids into peptide linkage diminished fluorescence markedly; at that time, the effect was ascribed to the strong electronegativity of the carbonyl group. Subsequent studies on solvent perturbation of fluorescence have verified the earlier experimental studies but have revealed that the fluorescence quenching is induced specifically by the carbonyl group rather than by electronegative groups in general. Because the carbonyl quenching and its relationship to general Biochim. Biophys. Acta, 133 (I967) 6-18
PEPTIDE
FLUORESCENCE
7
solvent effectsa, 4 are so important for the quantitative study of protein fluorescence, a reappraisal of these effects has been made and will be discussed in terms of their probable significance for fluorescence of tryptophanyl and tyrosyl residues in proteins. CHEMICALS
AND
METHODS
Chemicals Indole and phenol derivatives were obtained from commercial suppliers and identities were verified by melting point values. Peptides and other derivatives of tryptophan and tyrosine were purchased from Cyclo Chem. Co. and Mann Research Lab., Inc. Identities were verified by the fluorescence characteristics, by titration curves, and by paper chromatography. The A - a n d B-chains of insulin were supplied by the Eli Lilly Co. as the S-sulphonate derivatives. Angiotensin amide was supplied by Ciba Pharmaceutical Co. as Hypertension-Ciba powder (Lot B 5578). Organic solvents were purchased as spectro-grade quality and were tested for absorbing or fluorescing impurities. Where impurities were present, the solvent was distilled or (for the higher boiling solvents) treated with charcoal until free of the impurities. Peroxides in p-dioxane were removed by conventional treatment with SnCI., and distillation under N~. Methods Methods for measurement of pH, absorbance and fluorescence have been described earlierl, 5. The monochromators of the Aminco-Bowman spectrophotofluorometer were calibrated with a mercury arc lamp and the emission maxima were corrected for band width and variable sensitivity of the photomultiplier tube (IP28) by the method of PARKER AND REES 6. Values of emission maxima reported in Table I are considered accurate within ~-5 m/~. (The Coming filter CS 0-54 that was used with the spectrophotofluorometer in Paper I has been deleted in subsequent measurements of fluorescence.) Slight variance in light intensity from the xenon arc source was compensated by rapid successive readings of the sample and a reference standard (p-cresol or L-tyrosine at p H 7 for phenols; indole or L-tryptophan at p H 7 for indole derivatives). Quantum efficiency of fluorescence (Q) - - light quanta emitted as fluorescence per light quanta absorbed, and values were calculated by the method of TAI3I.E I VALUES
OF
EMISSION
MAXIMA
Emission peak maximum (my) (activation at 280 my)
5;olv e ~ l
;\:alz~l'e
Dielectric constant
Indole
Indole-3CH2CH2CN
N-aeetyltryptophan methyl ester
\Vater Methanol n-Propanol n-Butanol Butyl ether p-Dioxane n-Hexane
78.5 32.6 2o.1 17. t (4) z.2 1. 9
35 ° 330 33 ° 33 ° 3 l° 315 300
35 ° 34 ° 34 ° 34o 32o 325 3IO
35 ° 34 ° 34 ° 34o 33 ° 33 ° 3to
Biochim. Biophys. Acta, 133 (1967) 6 - i 8
R. W. COW(IlIA.
PARKER AND REES6 with the above-mentioned reference standards. (Preliminary tests showed that the ratio of height to area for the emission peak was constant for each of the phenol and indole series of compounds in all solvents; therefore, emission peak height was routinely measured.) Fluorometric titration procedures have been described in an earlier paper, and the present equations for fluorescence changes associated with ionization of specific groups are minor variations of equations derived in that paper 5. That is, Eqn. I: R E = RENH3 +
RENH2 - - RENH3
(~)
I -- antilog (pK'NH2- pHi
is a modification of Eqn. 6 of the earlier paper, in which R E = observed relative emission at any given pH, REs-H2 = fluorescence of the sample completely in the dissociated form and R E , H a = fluorescence of the sample completely in the protonated form. pK'NI-I2 is the dissociation constant under the conditions of the titration. Similarly, Eqn. 2 Rt2 = R E c o o H +
i
REcoo- -- RECOOH antilog (pK'cooH -- pH)
(2)
may be derived for the titration of the carboxyl group. Finally, Eqn. 3 is the same as RE= i
REo -7 antilog ( p H - pK'oH)
(3)
Eqn. 4 of the earlier paper for the titration of a phenolic group, for which the fluo rescent, unionized form (REo) passes to the non-fluorescent, ionized form with midpoint of the transition at pK'oH. RESULTS
Effects of the solvent It will be demonstrated that the solvent has a profound effect on the ability of a carbonyl substituent to quench the fluorescence of an indole or phenol compound. Therefore, initial consideration must be given to the general effects of the solvent on fluorescence. One effect that has been observed by a number of investigators3, 4 is a shift to lower wavelength for the emission peak of indoles when in solvents of non-polar character. Values in Table I show that this shift occurs both with indole and with the carbonyl-substituted derivative, N-acetyltryptophan methyl ester. Emission maxima at 305 m/z for the phenolic compounds listed in this paper did not vary (~: 5 m/x) when measured in the solvents of Table I. (Absorption spectra are not sensitive to variance of solvent for either the indole or phenol compoundsE) On the basis of these observations and the theory of LIPPERT for dipole orientation s, the indole ring must possess a higher dipole moment in the excited state than in the ground state. However, this does not seem to be directly related to the quenching phenomenon of carbonyl derivatives because the emission maximum shifted to about the same extent for the ester as for the parent compound. Also, the insensitivity to variation of solvent for the absorption and emission spectra of phenolic compounds, Biochim. Biophys. ;4cta, i33 (z967) 6 - i 8
PEPTIDE FLUORESCENCE
9
either with or without carbonyl substituents, would indicate that differences in dipole moment of the ground and excited states are minor for the latter compounds. Another effect of solvents on indole compounds is an increased fluorescence efficiency that generally occurs in solvents of low dielectric constantS, 4. Fig. I demonstrates this effect for water, a series of primary alcohols and two ethers. The upper two curves show that the effect on the fluorescence of indole and indole- 3propionitrile is small; in contrast, the lower two curves for the carbonyl-substituted derivatives indole-3-carboxylic acid and N-acetyltryptophan methyl ester show large increases of fluorescence for solvents of low dielectric constant. Similarly, the fluorescence output of anisole and phenol changed to only a slight extent when the polarity of the solvent was varied 9 but a striking rise in fluorescence, similar to that in Fig. I, occurs for carbonyl-substituted derivatives. In Fig. 2, the contrast is clear between i
J
i
12
i
34
i
u
i
5
6
7
%-,o~
x
i
0.4
0.3
0.2
i """-, x
~"
~'~
0. l L
I
I
40
20
-o
60
80
Dielectric Constant of the Solvent
Fig. I. Effect of the solvent on fluorescence output. Fluorescent compounds are indole ( ~ ) , indole-3-COOH ( O ), indole-3-(CH2)oCN ( • ) and N - a c e t y l t r y p t o p h a n m e t h y l ester ( × ). Solvents are (i) p-dioxane, (2) b u t y l ether, (3) n-butanol, (4) n propanol, (5) ethanol, (6) methanol and (7) water. i
i
i
i b
8.0
,2
.o
/
i
l c
/
x × /
i
I
- 4 , 0 ~ /
//
×J
- . x~i//I/ 1.0
/ I/ t
Xll l "llil
,1
i j
I
L
25 --5'0
A
as -50 z5 % (v/v) Organic Solvent Component
z5
50
~5
Fig. 2. Effect of water : organic solvent proportions on fluorescence. (a) Phenol (©) and N-acetyltyrosine amide ( × ) in w a t e r : d i o x a n e mixtures. (b) Indole ( 0 ) and N - a c e t y l t r y p t o p h a n m e t h y l ester ( × ) in water :dimethylsulfoxide mixtures. (c) Cresol ( 0 ) and N-acetyltyrosine ethyl ester ( × ) in water : polyethylenglycol-4oo mixtures.
Biochim.Biophys.Acta,I33 (I967)
6-I8
IO
R. ~,V. COWCILL
the fluorescence response of the parent indole and phenol compounds and that of the carbonyl derivatives to change of solvent composition. That is, the transition from aqueous solution to the various organic solvents led to modest increases in fluorescence of the parent compounds but much larger increases for the carbonyl derivatives. Fig. 3 expresses similar fluorescence changes in terms of the quantum efficiency of fluorescence (Q). Figs. I and 3 show that the fluorescence was much lower for carbonyl derivatives than for the parent compound in water but that they approach a common fluorescence efficiency in non-polar solvents. 1
1
i
I
o
0.4
J
J Q
J
S
J
o
J
J/ /
~
~
~o x
x//1 j j°
j
I
X'~
//
0.2
x
x xf t
j//
a
/
x I 20
I 40 %
I 60 (v/v)
I 80
Dioxone
Fig. 3- Effect of water : d i o x a n e s o l v e n t p r o p o r t i o n s on () for p-cresol ( © ), N - a c e t y l t y r o s i n e ethyl ester (CJ) a n d G l y - T y r - G l y - a m i d c ( × ) .
Effects of substituents Earlier studies in this laboratory1, 2 as well as subsequent studies in other laboratories1°, n on the quenching of fluorescence of tryptophan and tyrosine by unionized carboxyl groups and peptide bonds were limited to aqueous solutions. However, it now is apparent from the data in Figs. 1-3 that fluorescence efficiency of these carbonyl derivatives is the same as for the parent compounds when measured in non-polar organic solvents, and the earlier attempts to account for the fluorescence quenching must be reconsidered. A comparison of Q values for a variety of compounds is summarized in Table II, and the compounds have been divided into groups of high and low fluorescence in water. This division does not depend upon the electronegativity of the substituent since the -CN and -NH3+ groups are strongly electronegative, yet these derivatives are comparable to the parent compounds in fluorescence. Instead, the division depends upon the presence or absence of a carbonyl group in the side chain. Again it m a y be noted that in the non-polar solvent, p-dioxane, all of the indole compounds as well as those of the phenol series approach common levels of fluorescence. Therefore, the low fluorescence depends upon the presence of both a carbonyl substituent and a polar solvent that is able to solvate the carbonyl group b y hydrogen-bonding. (Note that dimethylformamide and dimethylsulfoxide, which are polar solvents but not donors for hydrogen-bonding, abolished the quenching phenomenon in the type of experiment shown in Fig. 2.) A series of carbonyl compounds were tested and the results in Table I I I indicate Biochim. Biophys. Acla, 133 (I967) 6-18
II
PEPTIDE FLUORESCENCE T A B L E II COMPARISON OF Q VALUES FOR A VARIETY OF COMPOUNDS
Fluorescent compound
Eyect of solvent on fluorescence efficiency (Q) 1Vater
n-Butanol
p-Dioxane
N - A c e t y l t r y p t o p h a n m e t h y l ester
o.II
0.26
0.53
Indole-3-CH2COOH
0.23
o.27
o.67
Indole
0.40
o.58
o.62
Indole-3-CH2CN
0.58
o.7o
o.58
Indole- 3-(CH2) aCN
0.59
0.72
0.62
Indole- 3 - (CH,,) 2NHa +
0.40
--
__
N - A c e t y l t y r o s i n e e t h y l ester
0.o6
o. I8
o.36
HO-~)
O.Ol
0.22
0.34
CH3Oq -CH2COOH
o.o2
0.30
o.31
HO-~>CH
0.23
o.39
0.45
o.16
o.34
o.34
CHaCOOH
3
HO-/~>CH2CN /--
TABLE III Q VALUES FOR CARBONYL COMPOUNDS
Fluorescent compound
Q in aqueous solution *
Tyrosine Tyrosine Tyrosine Tyrosine
o.21 o.o8 o.o6 o.o8
(CO0) (CONH2) (COOH) (COOCHa)
Tryptophan Tryptophan Tryptophan Tryptophan
(COO) (CONH2) (COOH) (COOCHa)
0.20 o. 14" * o.o9 o.o5**
I ndole-3-CH2COOIndole-3-CHzCONH 2 Indole-3-CH2COOH Indole-3-CH2COOCH a
0.45 * * 0.5 ° o.23 * * o.2I
* p H was c h o s e n on t h e basis of t i t r a t i o n curves s u c h as in Fig. 4 so t h a t a-NFI 2 groups were p r o t o n a t e d . * * Discrepancies b e t w e e n t h e s e v a l u e s a n d ones r e p o r t e d in P a p e r I {ref. I) arise f r o m i m p r o v e d choice of p H in t h e p r e s e n t e x p e r i m e n t s to e n s u r e c o m p l e t e p r o t o n a t i o n of t h e groups.
Biochim. Biophys. Acta, 133 (1967) 6 - 1 8
12
R. \V. COWGILL
that fluorescence quenching was strong for the acid, amide and ester but weak for the carboxylate ion. (Similar quenching was observed by FEITELSON for corresponding derivatives of tyrosine and phenylalaninel°.) The amide group appears to quench indole fluorescence to a smaller extent than do acid or ester groups, and this will be referred to later in connection with fluorescence of tryptophanyl peptides. A more marked contrast in the data of Table I I I is in the magnitude of these quenching effects on tryptophan fluorescence as compared with those for indole-3-acetic acid and its derivatives, even though the carbonyl group is separated from the indole ring b y a single methylene group in the latter case. This enhanced quenching of the tryptophan derivatives is attributed to the ~-amino group, and the action of the latter group has been further investigated. In conformity with the conclusion that
5.0
4.0
~
3.0
/X
2.0
/ fX ¢
~,
[.0
I
I
4
6
o -
I
I
8
I0
-~wOo
pH
Fig. 4, F l u o r o m e t r i c t i t r a t i o n s a t a c o n c e n t r a t i o n of 1. l o -6 M in a q u e o u s s o l u t i o n s a t 25 ~' a n d ionic s t r e n g t h o.i. ( R e l a t i v e e m i s s i o n v a l u e s were a d j u s t e d to t h e c o m m o n v a l u e of i.o a t p H 5-5 for c l a r i t y of t h e comparison,) T h e h e a v y lines r e p r e s e n t t h e o r e t i c a l r e l a t i o n s h i p s for fluorescence c h a n g e s a s s o c i a t e d w i t h i o n i z a t i o n of specific groups, a n d t h e s e were c a l c u l a t e d b y Eqns . i a n d 2 w i t h p K ' v a l u e s of 4.4 for indole-3-CH2CO2H (O) ; of 2. 4 a n d 9-3 for t r y p t o p h a n ( x ) ; a n d 7.5 for t r y p t o p h a n a m i d e (O). The final set of d a t a ( ~ ) is for t r y p t a m i n e .
the fluorescence quenching is not the result of a general inductive effect, the titration curve of tryptamine in Fig. 4 showed no change in fluorescence over the pH region for ionization of the amino group and the attendant change in its electronegativity. However, the fluorometric titrations of tryptophan and tryptophanamide did show a rise in fluorescence over the p H regions for ionization of the o-amino groups. (It m a y be noted that titration of indole-3-acetic acid into the alkaline region gave no change in fluorescence; therefore, the above-mentioned effects cannot be ascribed simply to an effect of OH-.) An enhancement of the fluorescence of tyrosyl peptides by dissociation of the c~-amino group also was indicated by data in Fig. 5 although interpretation of the evidence is complicated by ionization of the phenolic group. Fluorometric titration of tyramine gave no enhancement of fluorescence over the region for ionization of the amino group, and data for the p H region 8-1o.5 fits the theoretical curve for loss of fluorescence solely by the mono-basic titration of the phenolic group. Clearer evidence came from titration of Gly-Tyr-Gly-amide because Biochim. Biophys. Acta, ~33 (1967) 6-18
PEPTIDE FLUORESCENCE
13
ionization of the ~-amino group was displaced to a lower p H region. A significant rise in fluorescence occurred in the region for ionization of the ~-amino group and before the phenolic ring began to ionize. These fluorometric titrations, as well as the data in Table I I I , show that the amino group alone had no effect on fluorescence but that the e-NH 2 group and to even a greater extent the ~-NH3+ group enhanced the quenching of fluorescence b y the carbonyl group. The comparison of Q values in Table IV illustrates this effect in another fashion. These latter ionization effects are of less importance for fluorescence of proteins than the quenching action of the carbonyl group. However, the effects on fluorescence of the ionic state of neighboring carboxyl and amino groups is of considerable interest when fluorescence is applied to the study of the kinetics and the products of enzymatic i
i
i
I
;4 ×
×
x x
x
×
x x
IO
x
x
x
x
•
o _-__o
°°
o
x
.~ %x
\ 0.5
I
I
6
8
10
pH
Fig. 5. Fluorometric titrations of t y r a m i n e (samples from different sources, O and 0 ) and Gly T y r - G l y - a m i d e ( × ) . Titration conditions and a d j u s t m e n t of data to R E = i.o were as described in Fig. 4 except t h a t the concentration of fluorescent c o m p o u n d was 4" Jo-S M. The line represents the theoretical relationship, as expressed by Eqn. 3 if p K ' o ~ = 9.7, for titration of the phenolic group in the absence of a n y effect on fluorescence by ionization of the amino group. T A B L E IV
Fhtovescent compound
Q in aqueous solution
[ndole-3-CHaCH2-COO-
0.54
NH 2
I
11-.dole- 3-CH z C H - C O O -
o. 5 i
NHa+
[
I ndole-3-CH2CH-COO
0.20
Indole-3-CHiCH2COOH
0.37
NH~ +
I
Indole-3-CH2CH-COOH
o.o9
Biochim. Biophys. Acta, 133 (1967) ()-I8
14
R. \V. CO'~VC-ILL
hydrolysis of a protein. (Application of this technique to the model protein ribonuclease A has been fruitful and the results will be described in a later publication.) In anticipation of this application of fluorescence, some values of Q for tryptophanyl peptides not listed in the earlier papers are given in Tables V and VI. The arrangement of peptides in Table V in order of decreasing fluorescence confirms the observation in Table III that tryptophan fluorescence is less sensitive than that of tyrosine to an amide linkage on the a-carboxyl group. (For example, compare +Gly-Trp- and TABLE V
Fluorescent compound*
Q in aqueous solution
-Trp Leu+Trp-amide +Trp-Gly+Trp-Tyr+Trp-Leu-anlide +Leu-Trp +Leu-Trp-amide +Gly Trp
o.2 t o. 14 o. 14 o. 14 o. 1 i 0.09 o.o8 o.o5
* + and - signs indicate the ionic states of the :c-amino and carboxyl groups respectively. TABLE \:f
Fluorescent compound*
+Pro-Trp Pro~'rp+Gly-TrpGly T r p +Gly G l y - T r p Gly-Gly-Trp-
(pH 7.0) (pH lO.2) (pH 7.0)
(pH 9.8) (pH 7.0) (pH 9.8)
Q in aqueovts solution 0.05 o.19 0.05 O.14 0.o8 o.Io
* + and - signs indicate the ionic states of the a-amino and carboxyl groups respectively.
+Trp-Gly-; by contrast +Gly-Tyr and +Tyr-Gly gave the same value of Q2.) Note that the a-amino group still exerts a powerful effect even though separated from the fluorescent ring by one or more residues, and this is reasonable if the action of the a-amino group is to modify the electronegativity of the neighboring carbonyl group. Values for Pro-Trp in Table VI indicate that the positively charged nitrogen atom of proline also enhances the carbonyl quenching. Values for the Gly-Trp and Gly-Gly-Trp peptides show that the quenching effect levels off rapidly with an increase in the number of peptide bonds; similar observations have been made for peptides of tyrosine 1.
Nature of the combined effect of the solvent plus the carbonyl group on fluorescence quenching Energy of light quanta absorbed by the indole and phenol compounds would be released by the following pathways: (i) fluorescence, (2) degradation via external, Biochim. lRiophys. Acta, 133 (1967) 6 - i 8
PEPTIDE FLUORESCENCE
15
non-radiative processes, or (3) degradation via internal, non-radiative processes. (Passage through the phosphorescent triplet state m a y be considered as an additional pathway of degradation via internal, non-radiative processes in solutions at room temperature.) To the extent that the probability increased for energy dissipation by either pathway 2 or 3, the quantum efficiency of fluorescence would decrease. Hence, the fluorescence quenching noted above must be considered in terms of these two general paths for energy dissipation. A number of posibilities for energy dissipation via external, non-radiative processes were investigated. Non-specific solvent quenching does not appear probable because of the peculiar dependence of the quenching phenomenon upon the presence of carbonyl substituents. A more specific solvent quenching mechanism through formation of a solvent-cage with the carbonyl derivative does not seem probable on the basis of the following observations : (I) absence of consistent and significant differences in absorption spectra or emission spectra between the carbonyl derivatives versus the parent compounds (however, it is interesting that slight changes have been reported in the absorption spectra of tryptophan 1~ and tyrosine la upon ionization of the ~-amino and carboxyl groups) ; (2) absence of differences in effects on fluorescence of the carbonyl derivatives versus the parent compounds b y solvent-disruptive conditions such as temperature variation, detergents, or high concentrations of alcohols, urea or salts; (3) absence of the quenching effect with derivatives that contain other highly solvated substituents such as - N H 3 +, - O H or -COO-. Another possibility, that the carbonyl group need not be attached to the fluorescent compound in order to quench, was considered in an earlier paper 1. At that time, tests with aqueous solutions of acetic acid showed only quenching that might be expected from ionization of a fraction of the acid to acetate ions (the latter is effective for collisional quenching of phenolsg). More recently, FEITELSOX has reported quenching of fluorescence of tyrosine and phenylalanine by acetic and other carboxylic acids 1°. However, the possibility of quenching by the acid anions must be considered in these latter experiments also. To eliminate this uncertainty, tests were made in this laboratory for quenching by amides, esters, and peptides. No detectable quenching of the fluorescence of indole or phenol in aqueous solution was observed in the presence of as much as 2.5 M ethyl acetate (in 35% ethanol), o.57 M dimethylacetamide or o.I M alanylserine. (An optical arrangement was employed that permitted measurement from the front face of the cuvette to eliminate trivial absorption of activation and emission light.) Somewhat related to this question of quenching by free carboxyl compounds was the possibility that the carbonyl derivatives might dimerize, for this can result in loss of fluorescence of certain dyes 14. This possibility was excluded because dimerization should be less in aqueous solution than in the organic solvents and because the methyl ester of indole-3-acetic acid, which would not be expected to form a dimer in any solvent, did show the typical low fluorescence of the carbonyl derivatives. For all of these reasons, an external quenching mechanism does not seem probable. Energy degradation via some internal, non-radiative process is more probable because of the strict structural requirements; and several processes have been considered by FEITELSON1°, although he was unaware at that time of the importance of solvation and of the necessity that the carbonyl group be attached to the fluorescent molecule. Certainly a number of mechanisms could be considered for quenching by interconversion of electron energy levels between excited states of the types (singlet Biochim. Biopkys. Acla, 133 (1967) 6 18
I{)
R. \v. C()\VGILL
triplet) and (triplet-> triplet) for tim transitions ~ >.~* of the aromatic n - ~ = * of the carbonyl group. But it would be pointless to review them at for definitive experiments have not been made. Until such experiments are it will be assumed in these studies that the same internal quenching that observed with small peptides also will occur in the polypeptide chains of
ring and this time reported, has been proteins.
DISCUSSION
This discussion will be limited to the bearing that results of this study have on the fluorescence of tryptophanyl and tyrosyl residues in proteins. A number of attempts have been made to judge the environment of tryptophanyl residues in proteins from the wavelength of the emission peak. However, it m a y be noted in Table I that the correlation is poor between the wavelength m a x i m u m and dielectric constant of the solvent. (Even greater lack of correlation resulted when polarity of the solvent was gauged b y the dipole moment or the molar refractivity.) In view of this poor correlation and the uncertainty of the milieu in the protein interior, measurements of emission peaks appear to offer only a qualitative indication of the exposure of tryptophanyl residues to the aqueous solution. A more promising basis for judging the bonding and environment of fluorescent residues is the quantum efficiency of fluorescence. Results in this paper show that an exposed residue (Type ia) in an aqueous environment on the surface of the protein should be partially quenched by hydrated carbonyl groups of peptide bonds and carboxamide side-chains and should have a lower fluorescence than a buried residue (Type ib) in the non-aqueous interior of the molecule. Therefore, we can make a preliminary classification of these residues and an estimate of their probable fluorescence efficiency. An attempt in this direction already has been made for tyrosyl residues, and the designation of residues as Types I a and I b is in conformity with that classification system ~5. Values that are indicative of the Type i a residues on the surface of a protein are given in Table VII so that an assignment of a range of 0 = O.lO-O.15 for a Type I a tryptophanyl residue and of O = o.o5-o.o7 for a Type ia tyrosyl residue seems reasonable. (Undoubtedly the variation of values within these TABI.E VALUES PHANYL
\:[I OF
Q
AND
FOR
TYPE
TYROSYL
la
RESIDUES
FROM
STUDIES
OF
MODELS
\VITH
RF~SIDUES
Model compound
(2
Refereuce
o. 12-o. 15 0.08 o.I 4
i 1 a n d T a b l e V1
o.o 5 o.o6 0.035 o.o55 0.075 o.o5 ~
i5 i6 This laboratory This laborato@ Fig. 5
F o r tryptophan : Copolymers with glutamic acid or l y s i n e Small peptides
For lyrosil~e : [~educed r i b o n u c l e a s e A Oxytocin Insulin B-chain Angiotensin amidc Gly-Tyr-Gly-amide (uncharged)
B i o c h i m . B i o p h y s . Acta, I33 (1907) 6-I~;
FULLY
IgXPOSED
TRYPTO
PEPTIDE FLUORESCENCE
17
ranges reflect more subtle differences between residues.) Until we can define the property of water that confers the quenching capacity upon the carbonyl group, there will remain some uncertainty of this extrapolation to residues on the surface of proteins since the organization of water molecules on the protein surface m a y be quite different from bulk water. However, the failure of high salt concentrations, detergents, etc. to abolish the quenching effect in the model compounds would indicate that a specific ordered structure of the water was not essential. Values of Q for residues of Type lb that are buried in the hydrophobic interior of a protein are more uncertain. This is so because the nature of the protein interior, although of non-polar and hydrophobic characterlT, TM, is not well defined as a micro-environment; and because some non-polar solvents, such as butyl ether in Fig. I, can quench fluorescence for reasons that remain obscure. However, if we accept the data for dioxane in Table I I as representative of a non-quenching solvent of low polarity, then Type Ib residues should have 0 values in the range of o.5o-o.65 for tryptophan and o.3o-o.45 for tyrosine. Two possibilities for interference with this classification of Type ib residues must be considered. First, it is conceivable that the peptide bonds in the protein interior might interact through hydrogen-bonding to create the same electron distribution in the carbonyl groups as form such a favorable site for quenching in aqueous solutions. However, tests showed that no quenching of Leu-Trp amide or N-acetyltryptophan methyl ester in p-dioxane occurred in the presence of even 6 M N-methylacetamide. Second, residues in the interior still might be quenched by hydrated carbonyl groups on the surface. The high fluorescence (0 approx, o.5) of some proteins would suggest that this does not occur; also the data in Tables VI and V I I indicate that the quenching efficiency diminishes with distance. (Quenching probably will diminish as the sixth power of the distance between fluorescent ring and carbonyl group if energy dissipation is via the internal processes mentioned above.) The present paper is not the place to consider the fluorescence of specific proteins, but brief consideration should be given to the possible relevance of the above classification. Of proteins that contain tyrosine but no tryptophan, ribonuclease A has been the most thoroughly investigated and the present classification of residues has been helpful 1~. It has been necessary to extend the classification to three additional categories (Types I I - I V ) of tyrosyl residues that were extensively, if not completely, quenched in order to account for the low fluorescence of the native protein. In the case of proteins that contain both tyrosine and tryptophan, the major proportion of the fluorescence comes from the tryptophanyl residues. TEALE has measured the fluorescence of a large number of these proteins a and values of O vary ten-fold over the range o.o5-o.48. It is apparent that an assignment of the proportion of tryptophanyl residues that are either Type Ia or Ib would permit one arbitrarily to account for the fluorescence of proteins with values of () in the range of O.l-O.5; however, this classification would not account for the very low fluorescence of other proteins. Just as for the tyrosyl residues, it seems necessary to propose additional types of tryptophan residues that are strongly quenched in the protein. However, the quenching mechanisms that are effective for tyrosyl residues have been tested and found ineffective for the quenching of tryptophan residues. Among the possibilities that still merit consideration is the quenching of both tryptophanyl and tyrosyl residues by disulfide groups within the protein 19. I?iochim. Bi~phys. dcta, I33 (I9,57) 6 i8
I~
R. W. COWGILL
ACKNOWLEDGEMENTS
This research was supported by U.S. Public Health Service research grant GM IO-515-o 3 from the National Institute of General Medical Sciences, and the technical assistance of Mrs. JANE ADCOCK is deeply appreciated. REFERENCES 1 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 i8 19
W. COWGILL,Arch. Biochem. Biophys., lOO (1963) 36. W. COWGILL, Biochim. Biophys. Aeta, 75 (1963) 272W. J. TEALE, Biochem. J., 76 (196o) 381. L. VAN DUUREN, Chem. Rev., 63 (1963) 325. W. COWCILL, Biochim. Biophys. dcla, 94 (1965) 81. A. PARKER AND W. T. REES, Analyst, 85 (196o) 587 . YANARI AND F. A. BOVEY, J. Biol. Chem., 235 (196o) 2818. LIPPERT, Z. Eleclrochem., 61 (1957) 962. \VEBER AND K. ROSENHECK, Biopolymers Syrup., I (1964) 333. FEITELSON, J. Phys. Chem., 68 (1964) 391. D. FASMAN, E. BODENHEIMER AND A. PESCE, J . Biol. Chem., 241 (1966) 916. J. w DONOVAN, M. LASKO'~VSKI, JR. AND H. A. SCHERAGA,J. A ~ . Chem. Soc., 83 (1961) 2686. D. B. ~VETLAUFER, J. Y. EDSALL AND B. R. HOLLINGXVORTH,J. Biol. Chem., 233 (~958) 1421. E. RABINOWlTCH AND L. F. EPSTEIN, J. Am. Chem. Soc., 63 (1941) 69. R. ~V. COWGILL, Biochim. Biophys. dora, 12o (1966) 196. R. ~,V. COWGILL,Arch. Biochem. Biophys., lO 4 (1964) 84. L. STRYER, J . iVIol. Biol., 13 (1965) 482. M. PERUTZ, J. ]Viol. Biol., 13 (1965) 646. R. W. CO\VGILL,Biochim. Biophys. dcta, in t h e press. R. R. F. B. R. C. S. E. G. J. (;.
Biochim. Biophys. dcla, 133 (1967) 6-18
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 35005 FLUORESCENCE AND P R O T E I N STRUCTURE X. R E A P P R A I S A L OF SOLVENT AND STRUCTURAL EFFECTS ROBERT \¥. COX,VGILL The Bowman Gray School of Medicine, Wake Forest College, Winston-Salen~, N. C. ( U.S.A .)
(Received June 3rd, 1966)
SUMMARY I. Quantum efficiencies of fluorescence and emission maxima were measured in a number of solvents for tryptophanyl and tyrosyl peptides, as well as simpler derivatives of indole and phenol. Fluorescence was internally quenched by carbonyl groups but this occurred only when the molecule was solvated by a hydrogen-bonding solvent such as water or an alcohol. In non-polar organic solvents, quenching by the carbonyl group was lost and fluorescence rose to values of the parent indole and phenol compound. 2. Fluorescence quenching occurred with derivatives that contained unionized acid, amide, ester and peptide groups. These groups, when present on other solute molecules, did not quench; that is, external quenching of indole and phenol by amides, esters, etc., did not occur. An amino group alone had no effect on fluorescence, but in the free amino acids and peptides an c~-amino group enhanced the quenching action of a neighboring carbonyl group. Fluorometric titrations showed that this enhancement of the carbonyl quenching was in the order ~-NHa + > ~-NH 2 > H. 3. The bearing that these observations may have on fluorescence of proteins was discussed. A preliminary classification scheme for exposed and buried tryptophanyl and tyrosyl residues was described and a range of values was selected for the fluorescence efficiency of each type of residue.
INTRODUCTION One of the aims of this series of papers has been to define the conditions that affect the fluorescence output of tryptophanyl and tyrosyl residues so that predictions may be made of the chemical bonding and physical environment of these residues within or on the surface of a protein. The first two papers1, 2 demonstrated that the incorporation of these amino acids into peptide linkage diminished fluorescence markedly; at that time, the effect was ascribed to the strong electronegativity of the carbonyl group. Subsequent studies on solvent perturbation of fluorescence have verified the earlier experimental studies but have revealed that the fluorescence quenching is induced specifically by the carbonyl group rather than by electronegative groups in general. Because the carbonyl quenching and its relationship to general Biochim. Biophys. Acta, 133 (I967) 6-18
PEPTIDE
FLUORESCENCE
7
solvent effectsa, 4 are so important for the quantitative study of protein fluorescence, a reappraisal of these effects has been made and will be discussed in terms of their probable significance for fluorescence of tryptophanyl and tyrosyl residues in proteins. CHEMICALS
AND
METHODS
Chemicals Indole and phenol derivatives were obtained from commercial suppliers and identities were verified by melting point values. Peptides and other derivatives of tryptophan and tyrosine were purchased from Cyclo Chem. Co. and Mann Research Lab., Inc. Identities were verified by the fluorescence characteristics, by titration curves, and by paper chromatography. The A - a n d B-chains of insulin were supplied by the Eli Lilly Co. as the S-sulphonate derivatives. Angiotensin amide was supplied by Ciba Pharmaceutical Co. as Hypertension-Ciba powder (Lot B 5578). Organic solvents were purchased as spectro-grade quality and were tested for absorbing or fluorescing impurities. Where impurities were present, the solvent was distilled or (for the higher boiling solvents) treated with charcoal until free of the impurities. Peroxides in p-dioxane were removed by conventional treatment with SnCI., and distillation under N~. Methods Methods for measurement of pH, absorbance and fluorescence have been described earlierl, 5. The monochromators of the Aminco-Bowman spectrophotofluorometer were calibrated with a mercury arc lamp and the emission maxima were corrected for band width and variable sensitivity of the photomultiplier tube (IP28) by the method of PARKER AND REES 6. Values of emission maxima reported in Table I are considered accurate within ~-5 m/~. (The Coming filter CS 0-54 that was used with the spectrophotofluorometer in Paper I has been deleted in subsequent measurements of fluorescence.) Slight variance in light intensity from the xenon arc source was compensated by rapid successive readings of the sample and a reference standard (p-cresol or L-tyrosine at p H 7 for phenols; indole or L-tryptophan at p H 7 for indole derivatives). Quantum efficiency of fluorescence (Q) - - light quanta emitted as fluorescence per light quanta absorbed, and values were calculated by the method of TAI3I.E I VALUES
OF
EMISSION
MAXIMA
Emission peak maximum (my) (activation at 280 my)
5;olv e ~ l
;\:alz~l'e
Dielectric constant
Indole
Indole-3CH2CH2CN
N-aeetyltryptophan methyl ester
\Vater Methanol n-Propanol n-Butanol Butyl ether p-Dioxane n-Hexane
78.5 32.6 2o.1 17. t (4) z.2 1. 9
35 ° 330 33 ° 33 ° 3 l° 315 300
35 ° 34 ° 34 ° 34o 32o 325 3IO
35 ° 34 ° 34 ° 34o 33 ° 33 ° 3to
Biochim. Biophys. Acta, 133 (1967) 6 - i 8
R. W. COW(IlIA.
PARKER AND REES6 with the above-mentioned reference standards. (Preliminary tests showed that the ratio of height to area for the emission peak was constant for each of the phenol and indole series of compounds in all solvents; therefore, emission peak height was routinely measured.) Fluorometric titration procedures have been described in an earlier paper, and the present equations for fluorescence changes associated with ionization of specific groups are minor variations of equations derived in that paper 5. That is, Eqn. I: R E = RENH3 +
RENH2 - - RENH3
(~)
I -- antilog (pK'NH2- pHi
is a modification of Eqn. 6 of the earlier paper, in which R E = observed relative emission at any given pH, REs-H2 = fluorescence of the sample completely in the dissociated form and R E , H a = fluorescence of the sample completely in the protonated form. pK'NI-I2 is the dissociation constant under the conditions of the titration. Similarly, Eqn. 2 Rt2 = R E c o o H +
i
REcoo- -- RECOOH antilog (pK'cooH -- pH)
(2)
may be derived for the titration of the carboxyl group. Finally, Eqn. 3 is the same as RE= i
REo -7 antilog ( p H - pK'oH)
(3)
Eqn. 4 of the earlier paper for the titration of a phenolic group, for which the fluo rescent, unionized form (REo) passes to the non-fluorescent, ionized form with midpoint of the transition at pK'oH. RESULTS
Effects of the solvent It will be demonstrated that the solvent has a profound effect on the ability of a carbonyl substituent to quench the fluorescence of an indole or phenol compound. Therefore, initial consideration must be given to the general effects of the solvent on fluorescence. One effect that has been observed by a number of investigators3, 4 is a shift to lower wavelength for the emission peak of indoles when in solvents of non-polar character. Values in Table I show that this shift occurs both with indole and with the carbonyl-substituted derivative, N-acetyltryptophan methyl ester. Emission maxima at 305 m/z for the phenolic compounds listed in this paper did not vary (~: 5 m/x) when measured in the solvents of Table I. (Absorption spectra are not sensitive to variance of solvent for either the indole or phenol compoundsE) On the basis of these observations and the theory of LIPPERT for dipole orientation s, the indole ring must possess a higher dipole moment in the excited state than in the ground state. However, this does not seem to be directly related to the quenching phenomenon of carbonyl derivatives because the emission maximum shifted to about the same extent for the ester as for the parent compound. Also, the insensitivity to variation of solvent for the absorption and emission spectra of phenolic compounds, Biochim. Biophys. ;4cta, i33 (z967) 6 - i 8
PEPTIDE FLUORESCENCE
9
either with or without carbonyl substituents, would indicate that differences in dipole moment of the ground and excited states are minor for the latter compounds. Another effect of solvents on indole compounds is an increased fluorescence efficiency that generally occurs in solvents of low dielectric constantS, 4. Fig. I demonstrates this effect for water, a series of primary alcohols and two ethers. The upper two curves show that the effect on the fluorescence of indole and indole- 3propionitrile is small; in contrast, the lower two curves for the carbonyl-substituted derivatives indole-3-carboxylic acid and N-acetyltryptophan methyl ester show large increases of fluorescence for solvents of low dielectric constant. Similarly, the fluorescence output of anisole and phenol changed to only a slight extent when the polarity of the solvent was varied 9 but a striking rise in fluorescence, similar to that in Fig. I, occurs for carbonyl-substituted derivatives. In Fig. 2, the contrast is clear between i
J
i
12
i
34
i
u
i
5
6
7
%-,o~
x
i
0.4
0.3
0.2
i """-, x
~"
~'~
0. l L
I
I
40
20
-o
60
80
Dielectric Constant of the Solvent
Fig. I. Effect of the solvent on fluorescence output. Fluorescent compounds are indole ( ~ ) , indole-3-COOH ( O ), indole-3-(CH2)oCN ( • ) and N - a c e t y l t r y p t o p h a n m e t h y l ester ( × ). Solvents are (i) p-dioxane, (2) b u t y l ether, (3) n-butanol, (4) n propanol, (5) ethanol, (6) methanol and (7) water. i
i
i
i b
8.0
,2
.o
/
i
l c
/
x × /
i
I
- 4 , 0 ~ /
//
×J
- . x~i//I/ 1.0
/ I/ t
Xll l "llil
,1
i j
I
L
25 --5'0
A
as -50 z5 % (v/v) Organic Solvent Component
z5
50
~5
Fig. 2. Effect of water : organic solvent proportions on fluorescence. (a) Phenol (©) and N-acetyltyrosine amide ( × ) in w a t e r : d i o x a n e mixtures. (b) Indole ( 0 ) and N - a c e t y l t r y p t o p h a n m e t h y l ester ( × ) in water :dimethylsulfoxide mixtures. (c) Cresol ( 0 ) and N-acetyltyrosine ethyl ester ( × ) in water : polyethylenglycol-4oo mixtures.
Biochim.Biophys.Acta,I33 (I967)
6-I8
IO
R. ~,V. COWCILL
the fluorescence response of the parent indole and phenol compounds and that of the carbonyl derivatives to change of solvent composition. That is, the transition from aqueous solution to the various organic solvents led to modest increases in fluorescence of the parent compounds but much larger increases for the carbonyl derivatives. Fig. 3 expresses similar fluorescence changes in terms of the quantum efficiency of fluorescence (Q). Figs. I and 3 show that the fluorescence was much lower for carbonyl derivatives than for the parent compound in water but that they approach a common fluorescence efficiency in non-polar solvents. 1
1
i
I
o
0.4
J
J Q
J
S
J
o
J
J/ /
~
~
~o x
x//1 j j°
j
I
X'~
//
0.2
x
x xf t
j//
a
/
x I 20
I 40 %
I 60 (v/v)
I 80
Dioxone
Fig. 3- Effect of water : d i o x a n e s o l v e n t p r o p o r t i o n s on () for p-cresol ( © ), N - a c e t y l t y r o s i n e ethyl ester (CJ) a n d G l y - T y r - G l y - a m i d c ( × ) .
Effects of substituents Earlier studies in this laboratory1, 2 as well as subsequent studies in other laboratories1°, n on the quenching of fluorescence of tryptophan and tyrosine by unionized carboxyl groups and peptide bonds were limited to aqueous solutions. However, it now is apparent from the data in Figs. 1-3 that fluorescence efficiency of these carbonyl derivatives is the same as for the parent compounds when measured in non-polar organic solvents, and the earlier attempts to account for the fluorescence quenching must be reconsidered. A comparison of Q values for a variety of compounds is summarized in Table II, and the compounds have been divided into groups of high and low fluorescence in water. This division does not depend upon the electronegativity of the substituent since the -CN and -NH3+ groups are strongly electronegative, yet these derivatives are comparable to the parent compounds in fluorescence. Instead, the division depends upon the presence or absence of a carbonyl group in the side chain. Again it m a y be noted that in the non-polar solvent, p-dioxane, all of the indole compounds as well as those of the phenol series approach common levels of fluorescence. Therefore, the low fluorescence depends upon the presence of both a carbonyl substituent and a polar solvent that is able to solvate the carbonyl group b y hydrogen-bonding. (Note that dimethylformamide and dimethylsulfoxide, which are polar solvents but not donors for hydrogen-bonding, abolished the quenching phenomenon in the type of experiment shown in Fig. 2.) A series of carbonyl compounds were tested and the results in Table I I I indicate Biochim. Biophys. Acla, 133 (I967) 6-18
II
PEPTIDE FLUORESCENCE T A B L E II COMPARISON OF Q VALUES FOR A VARIETY OF COMPOUNDS
Fluorescent compound
Eyect of solvent on fluorescence efficiency (Q) 1Vater
n-Butanol
p-Dioxane
N - A c e t y l t r y p t o p h a n m e t h y l ester
o.II
0.26
0.53
Indole-3-CH2COOH
0.23
o.27
o.67
Indole
0.40
o.58
o.62
Indole-3-CH2CN
0.58
o.7o
o.58
Indole- 3-(CH2) aCN
0.59
0.72
0.62
Indole- 3 - (CH,,) 2NHa +
0.40
--
__
N - A c e t y l t y r o s i n e e t h y l ester
0.o6
o. I8
o.36
HO-~)
O.Ol
0.22
0.34
CH3Oq -CH2COOH
o.o2
0.30
o.31
HO-~>CH
0.23
o.39
0.45
o.16
o.34
o.34
CHaCOOH
3
HO-/~>CH2CN /--
TABLE III Q VALUES FOR CARBONYL COMPOUNDS
Fluorescent compound
Q in aqueous solution *
Tyrosine Tyrosine Tyrosine Tyrosine
o.21 o.o8 o.o6 o.o8
(CO0) (CONH2) (COOH) (COOCHa)
Tryptophan Tryptophan Tryptophan Tryptophan
(COO) (CONH2) (COOH) (COOCHa)
0.20 o. 14" * o.o9 o.o5**
I ndole-3-CH2COOIndole-3-CHzCONH 2 Indole-3-CH2COOH Indole-3-CH2COOCH a
0.45 * * 0.5 ° o.23 * * o.2I
* p H was c h o s e n on t h e basis of t i t r a t i o n curves s u c h as in Fig. 4 so t h a t a-NFI 2 groups were p r o t o n a t e d . * * Discrepancies b e t w e e n t h e s e v a l u e s a n d ones r e p o r t e d in P a p e r I {ref. I) arise f r o m i m p r o v e d choice of p H in t h e p r e s e n t e x p e r i m e n t s to e n s u r e c o m p l e t e p r o t o n a t i o n of t h e groups.
Biochim. Biophys. Acta, 133 (1967) 6 - 1 8
12
R. \V. COWGILL
that fluorescence quenching was strong for the acid, amide and ester but weak for the carboxylate ion. (Similar quenching was observed by FEITELSON for corresponding derivatives of tyrosine and phenylalaninel°.) The amide group appears to quench indole fluorescence to a smaller extent than do acid or ester groups, and this will be referred to later in connection with fluorescence of tryptophanyl peptides. A more marked contrast in the data of Table I I I is in the magnitude of these quenching effects on tryptophan fluorescence as compared with those for indole-3-acetic acid and its derivatives, even though the carbonyl group is separated from the indole ring b y a single methylene group in the latter case. This enhanced quenching of the tryptophan derivatives is attributed to the ~-amino group, and the action of the latter group has been further investigated. In conformity with the conclusion that
5.0
4.0
~
3.0
/X
2.0
/ fX ¢
~,
[.0
I
I
4
6
o -
I
I
8
I0
-~wOo
pH
Fig. 4, F l u o r o m e t r i c t i t r a t i o n s a t a c o n c e n t r a t i o n of 1. l o -6 M in a q u e o u s s o l u t i o n s a t 25 ~' a n d ionic s t r e n g t h o.i. ( R e l a t i v e e m i s s i o n v a l u e s were a d j u s t e d to t h e c o m m o n v a l u e of i.o a t p H 5-5 for c l a r i t y of t h e comparison,) T h e h e a v y lines r e p r e s e n t t h e o r e t i c a l r e l a t i o n s h i p s for fluorescence c h a n g e s a s s o c i a t e d w i t h i o n i z a t i o n of specific groups, a n d t h e s e were c a l c u l a t e d b y Eqns . i a n d 2 w i t h p K ' v a l u e s of 4.4 for indole-3-CH2CO2H (O) ; of 2. 4 a n d 9-3 for t r y p t o p h a n ( x ) ; a n d 7.5 for t r y p t o p h a n a m i d e (O). The final set of d a t a ( ~ ) is for t r y p t a m i n e .
the fluorescence quenching is not the result of a general inductive effect, the titration curve of tryptamine in Fig. 4 showed no change in fluorescence over the pH region for ionization of the amino group and the attendant change in its electronegativity. However, the fluorometric titrations of tryptophan and tryptophanamide did show a rise in fluorescence over the p H regions for ionization of the o-amino groups. (It m a y be noted that titration of indole-3-acetic acid into the alkaline region gave no change in fluorescence; therefore, the above-mentioned effects cannot be ascribed simply to an effect of OH-.) An enhancement of the fluorescence of tyrosyl peptides by dissociation of the c~-amino group also was indicated by data in Fig. 5 although interpretation of the evidence is complicated by ionization of the phenolic group. Fluorometric titration of tyramine gave no enhancement of fluorescence over the region for ionization of the amino group, and data for the p H region 8-1o.5 fits the theoretical curve for loss of fluorescence solely by the mono-basic titration of the phenolic group. Clearer evidence came from titration of Gly-Tyr-Gly-amide because Biochim. Biophys. Acta, ~33 (1967) 6-18
PEPTIDE FLUORESCENCE
13
ionization of the ~-amino group was displaced to a lower p H region. A significant rise in fluorescence occurred in the region for ionization of the ~-amino group and before the phenolic ring began to ionize. These fluorometric titrations, as well as the data in Table I I I , show that the amino group alone had no effect on fluorescence but that the e-NH 2 group and to even a greater extent the ~-NH3+ group enhanced the quenching of fluorescence b y the carbonyl group. The comparison of Q values in Table IV illustrates this effect in another fashion. These latter ionization effects are of less importance for fluorescence of proteins than the quenching action of the carbonyl group. However, the effects on fluorescence of the ionic state of neighboring carboxyl and amino groups is of considerable interest when fluorescence is applied to the study of the kinetics and the products of enzymatic i
i
i
I
;4 ×
×
x x
x
×
x x
IO
x
x
x
x
•
o _-__o
°°
o
x
.~ %x
\ 0.5
I
I
6
8
10
pH
Fig. 5. Fluorometric titrations of t y r a m i n e (samples from different sources, O and 0 ) and Gly T y r - G l y - a m i d e ( × ) . Titration conditions and a d j u s t m e n t of data to R E = i.o were as described in Fig. 4 except t h a t the concentration of fluorescent c o m p o u n d was 4" Jo-S M. The line represents the theoretical relationship, as expressed by Eqn. 3 if p K ' o ~ = 9.7, for titration of the phenolic group in the absence of a n y effect on fluorescence by ionization of the amino group. T A B L E IV
Fhtovescent compound
Q in aqueous solution
[ndole-3-CHaCH2-COO-
0.54
NH 2
I
11-.dole- 3-CH z C H - C O O -
o. 5 i
NHa+
[
I ndole-3-CH2CH-COO
0.20
Indole-3-CHiCH2COOH
0.37
NH~ +
I
Indole-3-CH2CH-COOH
o.o9
Biochim. Biophys. Acta, 133 (1967) ()-I8
14
R. \V. CO'~VC-ILL
hydrolysis of a protein. (Application of this technique to the model protein ribonuclease A has been fruitful and the results will be described in a later publication.) In anticipation of this application of fluorescence, some values of Q for tryptophanyl peptides not listed in the earlier papers are given in Tables V and VI. The arrangement of peptides in Table V in order of decreasing fluorescence confirms the observation in Table III that tryptophan fluorescence is less sensitive than that of tyrosine to an amide linkage on the a-carboxyl group. (For example, compare +Gly-Trp- and TABLE V
Fluorescent compound*
Q in aqueous solution
-Trp Leu+Trp-amide +Trp-Gly+Trp-Tyr+Trp-Leu-anlide +Leu-Trp +Leu-Trp-amide +Gly Trp
o.2 t o. 14 o. 14 o. 14 o. 1 i 0.09 o.o8 o.o5
* + and - signs indicate the ionic states of the :c-amino and carboxyl groups respectively. TABLE \:f
Fluorescent compound*
+Pro-Trp Pro~'rp+Gly-TrpGly T r p +Gly G l y - T r p Gly-Gly-Trp-
(pH 7.0) (pH lO.2) (pH 7.0)
(pH 9.8) (pH 7.0) (pH 9.8)
Q in aqueovts solution 0.05 o.19 0.05 O.14 0.o8 o.Io
* + and - signs indicate the ionic states of the a-amino and carboxyl groups respectively.
+Trp-Gly-; by contrast +Gly-Tyr and +Tyr-Gly gave the same value of Q2.) Note that the a-amino group still exerts a powerful effect even though separated from the fluorescent ring by one or more residues, and this is reasonable if the action of the a-amino group is to modify the electronegativity of the neighboring carbonyl group. Values for Pro-Trp in Table VI indicate that the positively charged nitrogen atom of proline also enhances the carbonyl quenching. Values for the Gly-Trp and Gly-Gly-Trp peptides show that the quenching effect levels off rapidly with an increase in the number of peptide bonds; similar observations have been made for peptides of tyrosine 1.
Nature of the combined effect of the solvent plus the carbonyl group on fluorescence quenching Energy of light quanta absorbed by the indole and phenol compounds would be released by the following pathways: (i) fluorescence, (2) degradation via external, Biochim. lRiophys. Acta, 133 (1967) 6 - i 8
PEPTIDE FLUORESCENCE
15
non-radiative processes, or (3) degradation via internal, non-radiative processes. (Passage through the phosphorescent triplet state m a y be considered as an additional pathway of degradation via internal, non-radiative processes in solutions at room temperature.) To the extent that the probability increased for energy dissipation by either pathway 2 or 3, the quantum efficiency of fluorescence would decrease. Hence, the fluorescence quenching noted above must be considered in terms of these two general paths for energy dissipation. A number of posibilities for energy dissipation via external, non-radiative processes were investigated. Non-specific solvent quenching does not appear probable because of the peculiar dependence of the quenching phenomenon upon the presence of carbonyl substituents. A more specific solvent quenching mechanism through formation of a solvent-cage with the carbonyl derivative does not seem probable on the basis of the following observations : (I) absence of consistent and significant differences in absorption spectra or emission spectra between the carbonyl derivatives versus the parent compounds (however, it is interesting that slight changes have been reported in the absorption spectra of tryptophan 1~ and tyrosine la upon ionization of the ~-amino and carboxyl groups) ; (2) absence of differences in effects on fluorescence of the carbonyl derivatives versus the parent compounds b y solvent-disruptive conditions such as temperature variation, detergents, or high concentrations of alcohols, urea or salts; (3) absence of the quenching effect with derivatives that contain other highly solvated substituents such as - N H 3 +, - O H or -COO-. Another possibility, that the carbonyl group need not be attached to the fluorescent compound in order to quench, was considered in an earlier paper 1. At that time, tests with aqueous solutions of acetic acid showed only quenching that might be expected from ionization of a fraction of the acid to acetate ions (the latter is effective for collisional quenching of phenolsg). More recently, FEITELSOX has reported quenching of fluorescence of tyrosine and phenylalanine by acetic and other carboxylic acids 1°. However, the possibility of quenching by the acid anions must be considered in these latter experiments also. To eliminate this uncertainty, tests were made in this laboratory for quenching by amides, esters, and peptides. No detectable quenching of the fluorescence of indole or phenol in aqueous solution was observed in the presence of as much as 2.5 M ethyl acetate (in 35% ethanol), o.57 M dimethylacetamide or o.I M alanylserine. (An optical arrangement was employed that permitted measurement from the front face of the cuvette to eliminate trivial absorption of activation and emission light.) Somewhat related to this question of quenching by free carboxyl compounds was the possibility that the carbonyl derivatives might dimerize, for this can result in loss of fluorescence of certain dyes 14. This possibility was excluded because dimerization should be less in aqueous solution than in the organic solvents and because the methyl ester of indole-3-acetic acid, which would not be expected to form a dimer in any solvent, did show the typical low fluorescence of the carbonyl derivatives. For all of these reasons, an external quenching mechanism does not seem probable. Energy degradation via some internal, non-radiative process is more probable because of the strict structural requirements; and several processes have been considered by FEITELSON1°, although he was unaware at that time of the importance of solvation and of the necessity that the carbonyl group be attached to the fluorescent molecule. Certainly a number of mechanisms could be considered for quenching by interconversion of electron energy levels between excited states of the types (singlet Biochim. Biopkys. Acla, 133 (1967) 6 18
I{)
R. \v. C()\VGILL
triplet) and (triplet-> triplet) for tim transitions ~ >.~* of the aromatic n - ~ = * of the carbonyl group. But it would be pointless to review them at for definitive experiments have not been made. Until such experiments are it will be assumed in these studies that the same internal quenching that observed with small peptides also will occur in the polypeptide chains of
ring and this time reported, has been proteins.
DISCUSSION
This discussion will be limited to the bearing that results of this study have on the fluorescence of tryptophanyl and tyrosyl residues in proteins. A number of attempts have been made to judge the environment of tryptophanyl residues in proteins from the wavelength of the emission peak. However, it m a y be noted in Table I that the correlation is poor between the wavelength m a x i m u m and dielectric constant of the solvent. (Even greater lack of correlation resulted when polarity of the solvent was gauged b y the dipole moment or the molar refractivity.) In view of this poor correlation and the uncertainty of the milieu in the protein interior, measurements of emission peaks appear to offer only a qualitative indication of the exposure of tryptophanyl residues to the aqueous solution. A more promising basis for judging the bonding and environment of fluorescent residues is the quantum efficiency of fluorescence. Results in this paper show that an exposed residue (Type ia) in an aqueous environment on the surface of the protein should be partially quenched by hydrated carbonyl groups of peptide bonds and carboxamide side-chains and should have a lower fluorescence than a buried residue (Type ib) in the non-aqueous interior of the molecule. Therefore, we can make a preliminary classification of these residues and an estimate of their probable fluorescence efficiency. An attempt in this direction already has been made for tyrosyl residues, and the designation of residues as Types I a and I b is in conformity with that classification system ~5. Values that are indicative of the Type i a residues on the surface of a protein are given in Table VII so that an assignment of a range of 0 = O.lO-O.15 for a Type I a tryptophanyl residue and of O = o.o5-o.o7 for a Type ia tyrosyl residue seems reasonable. (Undoubtedly the variation of values within these TABI.E VALUES PHANYL
\:[I OF
Q
AND
FOR
TYPE
TYROSYL
la
RESIDUES
FROM
STUDIES
OF
MODELS
\VITH
RF~SIDUES
Model compound
(2
Refereuce
o. 12-o. 15 0.08 o.I 4
i 1 a n d T a b l e V1
o.o 5 o.o6 0.035 o.o55 0.075 o.o5 ~
i5 i6 This laboratory This laborato@ Fig. 5
F o r tryptophan : Copolymers with glutamic acid or l y s i n e Small peptides
For lyrosil~e : [~educed r i b o n u c l e a s e A Oxytocin Insulin B-chain Angiotensin amidc Gly-Tyr-Gly-amide (uncharged)
B i o c h i m . B i o p h y s . Acta, I33 (1907) 6-I~;
FULLY
IgXPOSED
TRYPTO
PEPTIDE FLUORESCENCE
17
ranges reflect more subtle differences between residues.) Until we can define the property of water that confers the quenching capacity upon the carbonyl group, there will remain some uncertainty of this extrapolation to residues on the surface of proteins since the organization of water molecules on the protein surface m a y be quite different from bulk water. However, the failure of high salt concentrations, detergents, etc. to abolish the quenching effect in the model compounds would indicate that a specific ordered structure of the water was not essential. Values of Q for residues of Type lb that are buried in the hydrophobic interior of a protein are more uncertain. This is so because the nature of the protein interior, although of non-polar and hydrophobic characterlT, TM, is not well defined as a micro-environment; and because some non-polar solvents, such as butyl ether in Fig. I, can quench fluorescence for reasons that remain obscure. However, if we accept the data for dioxane in Table I I as representative of a non-quenching solvent of low polarity, then Type Ib residues should have 0 values in the range of o.5o-o.65 for tryptophan and o.3o-o.45 for tyrosine. Two possibilities for interference with this classification of Type ib residues must be considered. First, it is conceivable that the peptide bonds in the protein interior might interact through hydrogen-bonding to create the same electron distribution in the carbonyl groups as form such a favorable site for quenching in aqueous solutions. However, tests showed that no quenching of Leu-Trp amide or N-acetyltryptophan methyl ester in p-dioxane occurred in the presence of even 6 M N-methylacetamide. Second, residues in the interior still might be quenched by hydrated carbonyl groups on the surface. The high fluorescence (0 approx, o.5) of some proteins would suggest that this does not occur; also the data in Tables VI and V I I indicate that the quenching efficiency diminishes with distance. (Quenching probably will diminish as the sixth power of the distance between fluorescent ring and carbonyl group if energy dissipation is via the internal processes mentioned above.) The present paper is not the place to consider the fluorescence of specific proteins, but brief consideration should be given to the possible relevance of the above classification. Of proteins that contain tyrosine but no tryptophan, ribonuclease A has been the most thoroughly investigated and the present classification of residues has been helpful 1~. It has been necessary to extend the classification to three additional categories (Types I I - I V ) of tyrosyl residues that were extensively, if not completely, quenched in order to account for the low fluorescence of the native protein. In the case of proteins that contain both tyrosine and tryptophan, the major proportion of the fluorescence comes from the tryptophanyl residues. TEALE has measured the fluorescence of a large number of these proteins a and values of O vary ten-fold over the range o.o5-o.48. It is apparent that an assignment of the proportion of tryptophanyl residues that are either Type Ia or Ib would permit one arbitrarily to account for the fluorescence of proteins with values of () in the range of O.l-O.5; however, this classification would not account for the very low fluorescence of other proteins. Just as for the tyrosyl residues, it seems necessary to propose additional types of tryptophan residues that are strongly quenched in the protein. However, the quenching mechanisms that are effective for tyrosyl residues have been tested and found ineffective for the quenching of tryptophan residues. Among the possibilities that still merit consideration is the quenching of both tryptophanyl and tyrosyl residues by disulfide groups within the protein 19. I?iochim. Bi~phys. dcta, I33 (I9,57) 6 i8
I~
R. W. COWGILL
ACKNOWLEDGEMENTS
This research was supported by U.S. Public Health Service research grant GM IO-515-o 3 from the National Institute of General Medical Sciences, and the technical assistance of Mrs. JANE ADCOCK is deeply appreciated. REFERENCES 1 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 i8 19
W. COWGILL,Arch. Biochem. Biophys., lOO (1963) 36. W. COWGILL, Biochim. Biophys. Aeta, 75 (1963) 272W. J. TEALE, Biochem. J., 76 (196o) 381. L. VAN DUUREN, Chem. Rev., 63 (1963) 325. W. COWCILL, Biochim. Biophys. dcla, 94 (1965) 81. A. PARKER AND W. T. REES, Analyst, 85 (196o) 587 . YANARI AND F. A. BOVEY, J. Biol. Chem., 235 (196o) 2818. LIPPERT, Z. Eleclrochem., 61 (1957) 962. \VEBER AND K. ROSENHECK, Biopolymers Syrup., I (1964) 333. FEITELSON, J. Phys. Chem., 68 (1964) 391. D. FASMAN, E. BODENHEIMER AND A. PESCE, J . Biol. Chem., 241 (1966) 916. J. w DONOVAN, M. LASKO'~VSKI, JR. AND H. A. SCHERAGA,J. A ~ . Chem. Soc., 83 (1961) 2686. D. B. ~VETLAUFER, J. Y. EDSALL AND B. R. HOLLINGXVORTH,J. Biol. Chem., 233 (~958) 1421. E. RABINOWlTCH AND L. F. EPSTEIN, J. Am. Chem. Soc., 63 (1941) 69. R. ~V. COWGILL, Biochim. Biophys. dora, 12o (1966) 196. R. ~,V. COWGILL,Arch. Biochem. Biophys., lO 4 (1964) 84. L. STRYER, J . iVIol. Biol., 13 (1965) 482. M. PERUTZ, J. ]Viol. Biol., 13 (1965) 646. R. W. CO\VGILL,Biochim. Biophys. dcta, in t h e press. R. R. F. B. R. C. S. E. G. J. (;.
Biochim. Biophys. dcla, 133 (1967) 6-18