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Fluorescence and the structure of proteins. I. Effects of substituents on the fluorescence of indole and phenol compounds
ARCHIVES
OF
BIOCHEMISTRY
Fluorescence
AND
BIOPHYSICS
and the Structure
on the Fluorescence
of Proteins.
of lndole
ROBERT From the Department
36-44 (1963)
I@,
of Biochemistry,
and
I. Effects of Substituents Phenol
W. COWGILL’, University Colorado
Received
of Colorado
August
Compounds
* School of Medicine,
Denver,
3, 1962
The quantum efficiency of fluorescence was measured for simple derivatives of indole and phenol and for tryptophyl and tyrosyl residues in peptides and proteins. Fluorescence of these compounds decreased as the electronegativity of substituents increased. In proteins, one strongly electronegative structure is the peptide bond. It is proposed that the observed, low fluorescence of tryptophyl and tyrosyl residues in proteins is attributable in large measure to the presence of this bond. INTRODUCTION
of tyrosine and tryptophan and ascribed losses in fluorescence to quenching by hydrogen ions, hydroxyl ions, and ionization of the phenolic hydroxyl group. The present study clearly reveals the high sensitivity of the fluorescence of both the indole and phenol rings to the electronegativity of substituents. In agreement with the earlier observations of Duggan et al. (4), increased electronegativity of substituents results in decreased fluorescence, and most of the observations of White (5) are explicable on this basis. One of the most powerful electronegative structures in proteins is the peptide bond, and suppression of fluorescence by the peptide chain is sufficient to account for the low fluorescence of some proteins.
The indole and phenol rings of tryptophan and tyrosine are fluorescent in proteins (1, 2). However, the quantum efficiency of fluorescence per tryptophyl or tyrosyl residue is not the same for all proteins (3) and is not even the same for different tryptophyl or tyrosyl residues in the protein molecule.3 This laboratory is now seeking the causes of these differences. The guiding hypothesis will be that the differences arise from variations in the local environment of the residues within or on the surface of the protein molecule, and the present paper is concerned with the inductive effects of groups near the aromatic ring. Duggan et al. (4) observed a loss of fluorescence upon acetylation of phenols and concluded that the fluorescence of the benzene ring depended upon the presence of a substituent (hydroxyl or amino group) able to donate electrons to the ring. White (5) studied the effect of pH on the fluorescence of peptides and other derivatives
MATERIALS
AND
METHODS
CHEMICALS Compounds for this study were obtained from sources indicated in Tables I and II. Identity and purity of the simpler compounds were established by absorption spectra, by fluorescence excitation and emission spectra, and by paper chromatography. Purification by distillation or crystallization was done when necessary. Identity and purity of peptides and other derivatives of tyrosine and tryptophan were established by the above tests as well as by hydrolysis to the amino acids. The latter were identified by paper chromatography. Insulin
1 Present address, Department of Biochemistry, Bowman Gray School of Medicine, Winston Salem, North Carolina. 2 This research was done during the tenure of a Research Career Development Award of The National Institutes of Health. 3 Unpublished work of this laboratory. 36
FLUORESCENCE
OF INDOLE
(tryst. Zn insulin, lot Xo. 7191Oti of Eli Lilly & Co.), pancreatic ribonuclease (chromatographitally pure, Type III, lot No. R22B-70 of Sigma trypsin inhibitor Chemical Co.), pancreatic (crystalline, lot, No. 1037 of Mann Research Laboratory), and zein (lot No. B2901 of Mann Research Laboratory) were used without purifical,ioll. ~~EASUREMENT
OF I~U~IWSCEIYCE
Fluorescence was measured in an AmincoBowman spectrophotofluorometer fitted with a 150-w. xenon lamp, slit. xsrangement I?r’o.3 and a Corning filter CS O-54 The filter removed scatter below 310 mp. Excitation and emission monochromators were calibrated with a mercury lamp. Fluorescence emission spectra were recorded on a chart recorder, and areas under the emission curves were determined by planimetry. Over the period of these measurements, the intensity of the xenon lamp varied because of variations in line voltage and the increasing age of the lamp. Since a comparison of quantum efficiency of fluorescence is valid only for constant light intensity, a correction was made in all measurements of fluorescence. The fluorescence of a reference solution (i nlg. tyramine. HCl/500 ml. water) was read at the same time as the measurement of fluorescence of a sample, and all readings were adjusted to a constant relative emission (R.E. = 1.98) for the t)-ramine reference solution. Fluorescence was measured in a concentration range t,hat ensured a linear relationship between fluorescence emission and concentration of the indole (lo-jlo* .&f) or phenol (10-4-10-” h1) compound. CALCULATION OF &IYAKTUM EFFICIBXCP 0~ FLUORESCXNCE Quant’urn efficiency of fluorescence was calculated by the method of Parker and Rees (6). This is a comparative method in which it is assumed that for a given spectrophotofluorometer the integrated area (A) under the fluorescence emission curve is proportional to the tot,al intensity of fluorescent light (F) emitted by the solution. The latter value (F) in turn is proportional to the where lo = intensity of excit.. product Za.Q.E.c.d, ing light, Q = quantum efficiency of fluorescence,4 E = molecular extinction coefficient, c = concent,ration, and d = optical path length. For comparison of fluorescence in t,he same apparatus and 4 The symbol Q is employed for quantum efficiency of fluorescence rather t,han t.he symbol + employed by Parker and Rees. The latter symbol has been employed for some time to denote energy yield of fluorescence [see Ref. (7), p. 3131.
8T
AND PHENOL
at the same intensity of excitation light, the ratio of fluorescence intensities is given by the equation:
F,
-z-z s;
A, A:
Q,.E,.c,.dl Qc.E2.cr.d?
= ($)(E$)
(l)
or
(2) Values for optical density at the wavelength of excitation and of the area under the observed emission spectral curve were determined for each compound. In order to avoid the necessity of COPrecting observed emission spectra for variation in intensity of the xenon lamp with wavelength of excitation, the exciting light was maintained constant at 280 rnr for all compounds. A second factor that may influence the emission spectrum is variation in t.he sensitivity of the photomultiplier tube to light of different wavelengths. Over the range of 300-400 rnp employed in this study, the RCA photomultiplier t,ube No. 11’ 28 is reported to vary in sensitivity of response by only 105, (8). This variation and any variat,ion in transmission of the emission monochromator have been neglected in measurement.s of areas under emission spectral curves.s. 6 Ratios of 3/O.D. for compounds bearing the indole ring and the phenol ring were compared with the values for tryptophan and tyrosine, respectively. Quantum efficiencies were calculated by Eq. (2) employing the reference values for Q of 0.20for t,ryptophan and 0.21 for tyrosine at neutral pH as reported by Teale and Weber (2). Values 6 Parker and Rees (6) have suggested that comparison of solutions may be based on intensity of fluorescence at the maximum of the emission curve once it has been established that, the ratio of area to spectral peak height is constant. This ratio is constant for hot.11 the tyrosine and tryptophan compounds of Tables IV and VI. However, the author prefers to rely on the area as measured by a planimeter since the ratio of area to peak height for tyrosine compounds in Table IV was found to vary from that for phenol compounds in Table III. Variations in this mtio have also been observed for f.ryptophan and tyrosine derivatives in other solvents and for tryptophyl and tyrosyl residues in different proteins. 6 In calculation of Q, t,he error from disregarding variations in the sensitivity of t,he photomult,iplier tube and in the transmission of the emission monochromator is estimated to be less t.han 5%. This error is small because t.he fluorescence spectral peaks are sharp and the fluorescence maxima are similar for compounds of both the indole and phenol t,ypes.
38
COWGILL TABLE
OPTICAL
ABSORPTION
AND
FLUORESCENCE
n-Tryptophan N-Ac-n-tryptophan rn-Tryptophan amide N-Ac-r-tryptophan methyl ester n-Tryptophan ethyl ester Glycyl-n-tryptophan Indole-3carboxylic acid Indole-3-acetic acid Indole
I
CHARACTERISTICS
OF INDOLE
AND
RELATED
COMPOUNDS”
278 280 278 -
6000 5900 5500 -
282 282 282 282
355 355 355 355
Mann Res. Lab. Lot No. A 5038 Mann Res. Lab. Lot No. A 4821 Sigma Chem. Co. Lot No. T6OB-061 HM Chem. Co. Lot No. 113-100-19
278 280 278 279 278
6000 5800 8400 7000 5606
282 282 282 282 282
355 355 360 365 355
Mann Res. Lab. Lot No. B 1978 Mann Res. Lab. Lot No. 1438 K & K Labs. Lot No. 31518 K & K Labs. Lot No. 20988 Fisher Sci. Co. Lot No. 710800
QX, and +, are respectively the optical absorption maximum in millimicrons and the molar extinction coefficient in aqueous solution at pH 7. Neither acidity not basicity of the solution affected the spectra of the indole compounds markedly. Acidity of the solution did not affect the spectra of the A, and Em are the wavelength maxima in millimicrons of the fluorescence phenol compounds markedly. activation and fluorescence emission spectra, respectively. These latter values have not been corrected for idiosyncrasies of either the xenon lamp or the IP28 photocell of the spectrophotofluorometer. TABLE OPTICAL
ABSORPTION
AND
FLUORESCENCE
II
CHARACTERISTICS
OF PHENOL
AND
RELATED
COMPOUNDS~
Compound
m-Cresol m-OH benzyl alcohol Phenol m-OH benzoic acid m-OH benzaldehyde
270 272 268 287 311
1750 1600 1500 2100 2700
279 280 278 295 -
320 320 320 413 -
K & K Labs. K & K Labs. Mallinckrodt K & K Labs. K & K Labs.
nn-Tyrosine n-Tyrosylglycine Glycyl-n-tyrosine Tyramine Glycyl-n-tyrosylglycinamide Leucyl-n-tyrosine
274 274 274 274 274
1520 1460 1280 1480 1480
285 285 285 285 285
320 320 320 320 320
Nutritional Biochem Mann Research Labs. Mann Research Labs. Mann Research Labs. Mann Research Labs.
274
1360
285
320
Mann
(1See footnote
to Table
calculated in this fashion reported in the literature Table VIII. RESULTS
AND
Research
Lot Lot USP Lot Lot
No. 10429 No. 31850 Cryst ., “Gilt No. 15657L No. 3072OL Co. Lot Lot Lot Lot
Lot No. No. No. No.
Label”
No. 8060 C 1054 D 4280 CA 2841 C 1151
Labs. Lot No. E 2273
I. compare well with those (3, 5), for example, see DISCUSSION
The compounds for this study are listed in Tables I and II with values for optical absorption maxima, molar extinction coefficients, fluorescence excitation maxima, and emission maxima. It may be observed that these values are similar for the different compounds of both the indole and the phenol series. Therefore, the nature of the
substituent has no appreciable effect on the excitation or emission maxima.7 The change which does occur with change of structure is in the intensity of fluorescence, that is, the quantum efficiency (Q) of fluorescence. The term (Q) may be defined as the ratio of the number of quanta of light emitted as fluorescence/number of quanta of light absorbed by the fluorescent substance. Values for Q have been determined by the com7 One exception in this benzoic acid of Table II.
series
is nL-hydroxy-
FLUORESCENCE TABLE QUANTUM
III
I~FFICIENCY (CJ) OF FIXORESCENCE META-SUBSTITTITED PHENOLS
-CHa -CH,OH
-H -coo-COOH
-CHO
OF INI~OLE
FOR
0.25 0.25 0.21 0.05 0.01
< 0.01
parative method described in the preceding section. COMPOUNDS THAT CONTAIN PHENOL RIKG
THE
The marked effects on the quantum efficiency of fluorescence by substituents introduced directly on the phenolic ring are shown in Table III. The loss of fluorescence is in the order to be expected on the premise that substituents of increasing electronegativity (that is, substituents that have an increasing ability to pull electrons from the ring) lead to decreasing fluorescence. Derivatives with hydroxymethyl or carboxaldehyde substituents at the ortho or para positions gave the same Q values as did the corresponding meta derivatives in Table III. So the position of the substituent on the benzene ring had no influence on the fluorescence of these compounds.R The phenolic rings of tyrosine and peptides containing tyrosine also show loss of fluorescence with the introduction of increasingly electronegative groups. The principal electronegative groups in these compounds have been underscored in Table IV. In these compounds, the inductive effect is attenuated by t.he intervening side chain so that the loss of fluorescence is not as marked as for compounds in Table III. There are 8 This conclusion does not apply to isomers in which the absorption spectrum is different for the various ring positions of substituents; for example, the hydroxpbenzoie acids.
AND
39
I’HENOI,
indications in Table IV that factors other than electronegativity influence the fluorescence of tyrosine in peptides, for example, compare leucyltyrosine (Q = 0.103) and glycyltyrosine (Q = 0.070). The influence of various amino acid side chains on the quantum efficiency of fluorescence will be considered in a second paper. COMPOUNDS THAT COSTAIN INJDOLX Rrsc,
THE
Efiects of electronegative substituents on the quantum efficiency of fluorescence of the indole ring are shown in Table V. The loss of fluorescence is greater for a -OH group than for the -COOform as would be expected from the greater electron-attracting power of the former group. Also, the attenuat,ion of the effect, by the interposition of even one methylene group between t,he electronegative substituent and the ring is clear. The effect of an electronegative group is further attenuated in peptides and other derivatives of tryptophan listed in Table VI. For example, compare indole-COO(Q = 0.24), indole-CHzCOO- (Q = 0.38), and indole-CHzCH(XH,)-COO(Q = 0.51). A carboxyl group in the solution but not a part of the fluorescent molecule has no comparable action on the fluorescence of either the indole or phenol ring. Both acetic acid and acetate ions weakly quench the fluorescence of tyrosine. The data conform with the Stern-Volmer equation for fluorescence quenching, and concentrations for half-quenching are 0.8 M acetic acid and 0.15 11f sodium acetate. Similarly, 0.8 Al acetic acid is required t,o half-quench tryptophan, but sodium acetate shows no quenching of tryptophan even at 0.4 A/. Quenching of tyrosine by other carboxylic acids in the same concentration range (0.1-0.8 M) has been reported by Teale (3). Certainly this quenching effect requires concentrations of the carboxylic acid several orders of magnitude higher than the concentration of compounds in Tables III-VI. Also, there is other evidence that such quenching is quite unrelated to the inductive effect of carboxyl groups attached to the fluorescent molecule. For example,
40
COWGILL TABLE QUANTUM EFFICIENCY
OF
IV
FLUORESCENCE FOR TYROSINE HO-’
0
PEPTIDES
AND
CONTAININGTYROSINE
’ -CHzCH-RI
-
Compound
RX
R2
RI
Tyrosine
-coo-
Tyramine
-H
HsN+H&+-
Leu-Tyr
--coo-
H~N+-cH--C~--?~TH-
-CO--SH-CH,-COO-coo-
Tyr-Gly Gly-Tyr
I Leu
0.103
-
H.N+-
0.074
HaN+-CHs-CO-NH-
0.070
HaN+-
0.056 0.050
-CO--NH-CH?-a-NH2 -CZIH
H3N+-CHy-CO-NH-
0.027
Gly-Tyr-glycinamide Gly-Tyr
V
QUANTUMEFFICIENCYOF FLTJORESCENCE FOR INDOLE COMPOUNDS
-H -CH&OO-coo-CH&OOH -COOK
0.185
-
-COOH AX--NH-CH,-COOH
TABLE
0.21
HsN+Hd+-CHz-CO-NH-
Tyr-Gly
Tyrosine
Q
0.40 0.38 0.24 0.16 0.08
the acetate ion is more effective than acetic acid for quenching tyrosine while the -COOH group is more effective than the -COOionic form for decreasing the fluorescence of the compounds in Tables III-VI. Also, the acetate ion has no quenching effect on tryptophan while the attached -COOgroup has a comparable effect on both the indole and phenol compounds of Tables III-VI. EFFECTS OF pH AND BUFFERS ON FLUORESCENCE Fluorescence was measured in aqueous solutions that were adjusted to insure the
-
0.035
presence of the desired ionic species of the fluorescent compound. For this purpose, measurements were made in 0.01 N HCl, 0.05 M Tris buffer of pH 7.5 for the phenol compounds, and in 0.005 M phosphate buffer of pH 6.0 or 10.5 for the indole compounds. White (5) has reported quenching of the fluorescence of tyrosine and tryptophan derivatives in hydrochloric acid. In the present studies, no marked quenching effect of WC1 on the fluorescence of phenol compounds was noted although the fluorescence of indole compounds is more sensitive to this acid (Fig. 1). It would appear that some of the changes in fluorescence with change in pH, which White ascribed to quenching by hydrogen ions, were actually due to changes in ionic species of the compound. Figure 2 shows the effect of pH on the quantum efficiency of fluorescence of tyrosine and tryptophan. These curves are similar in shape and points of inflection to curves given by White. 1% will be observed in Fig. 2 that the quantum efficiency of fluorescence is constant for both tyrosine and tryptophan in the region of pH 4-8. In more acidic solutions the Q values fall for both amino acids, and the pH at the midpoints of these decreases
FLUORESCENCE
OF INDOLE
ANI)
PHENOL
41
TABLE VI QUANTUM
OF FI,.L;ORESCENCE FOR
EFFICIXNCY
Compound
0.51
-coo-
CH,-CO-NH-
0.28
-CJ
H&-
0.28
-coo-
H$+-
0.20
-co- NH? -COOH -
H 2 U+-
0.20
CH&SO-NHHa?U’+-
0.128 0.080
WAC-tryptophan nmidc
Tryptophnn amide
K-Ac-tryptophan
Q
HZN--
-coo-
Tryptophan
RZ
RI
Tryptophnn Tryptophnn
NH?
0.085
F-AC Me ester of tryptophan
-COOH -COOCH3 -
EC ester of tryptophan
-COOC2Hs -
CH,-CO-NHH,N-
El ester of tryptophan
-COOCrHj -
H3?1;+-
0.032
Gly-Try
-coo-
0.095
Gly-Try
-coo-
H?S-CHT-CO-SHHay+-CHr-CO-NH-
0.057
Gly-Tr)
-coo1
&S+-CHZ-CO-XH-
0.022
TrZ’ptopllall
:. E To
TRYPTOPHAN, ITS PEPTIDES AND OTHERI)ERIvATIvEs
--...-..-.
-..._.-
.-.-.-.-.a
.-.-._.
0.076
-.-.m.
3..\ -,:““-,--
E
--------..----------_,,__
-*---mm.
..\
.t
.
A., .
.
. y-1.
0 I-
.---_ ---__
.
FIG. 1. Effect of hydrochloric acid on the fluorescence of peptides of tyrosine and tryptophan. Tyrosylglycine (1 X 1OP M) ----; Leucyltyrosine (1 X IOV J!f) - - - - -; GlJwyltyrosine (1 X lo-” ,!I) - - - - - - -; J,eucyltryptophan (I X lo-” Jf) - - -- -; Blycylt.ryptophan (1 x lo-j!ll) . . .
correspond to the dissociation constants (9) of the carboxyl groups (for tyrosine, pK1 = 2.20; for tryptophan, pK1 = 2.38). Similarly, changes in the alkaline region of pH 8-11 can be explained by changes in ionization. As mentioned by White, the fall in fluo-
Frc:. 2. Effect of pH on the quantum efficiency of fluorescence. Tyrosine -n-n--; Tryptophan -O-O-; Methyl ester of S-acetyltryptophan --O--O-. (No other hufer was added.)
rcscence of tyrosine occurs with a midpoint at pH 10.0 which compares well with its pKa = 10.06 (9) for ionization of the hydroxyl group and confirms previous observations that the ionized phenolic ring is nonfluorescent for tyrosine and its derivatives. The rise in fluorescence of tryptophan with a midpoint at pH 9.5 corresponds to
42
COWGILL
the dissociation of a proton from the amino group with p& = 9.39 (9). (Conversion of a -NH,+ group to a less electronegative --NH2 group would be expected to increase the fluorescence of the indole ring.) Beyond pH 11, tryptophan also loses fluorescence ; and this is presumably due to the quenching action of the hydroxyl ion as described by White. The proposal that changes in fluorescence in the regions of pH 13 and pH 8-11 are due to changes in ionic structure, is substantiated by the curve for the methyl ester of acetyltryptophan in Fig. 2. This ester displays neither the fall in fluorescence at low pH nor the rise in the region of pH 8-11. These differences from the curve for tryptophan are to be expected since the carboxyl group is esterified and the amino group is acetylated so that the molecule does not undergo change of structure with change of pH. Figure 2 should serve to emphasize the fact that values for & in Tables III-VI are approximations for those structural forms which exist only at high acidity or alkalinity. This is so because conditions chosen for measurement in 0.01 N HCl or in buffer at pH 10.5 do not necessarily correspond in all cases to minima or maxima of curves similar to those in Fig. 2. Tris buffer at 0.05 M and pH 7.5 showed no quenching effects with either indole or phenol compounds. This buffer was used in preference to phosphate for the phenol
compounds because fluorescence of the latter was strongly quenched by phosphate. As may be observed in Pig. 3, the HP04-ionic species is more effective than H,PO,for quenching fluorescence of phenol. The fluorescence of indole is weakly quenched by phosphate, but the more effective ionic species is H2P04- rather than HPO,-. RELATIONSHIP BETWEEN THESE OBSERVATIONS .4m THE FL~ORERCEY~CE OF TRYPTOPHAK AND TYROSINE IN
PROTEINS
The bearing that observations made in this paper have on fluorescence of proteins must be considered from two aspects. One is the effect of the peptide chain itself; the second is the effect of electronegative amino acid residues along the pept.ide chain. The effect of increasing peptide chain TABLE
VII
DECREASE IN FLUORESCENCE OF TYROSYL TRYPTOPHYL RESIDUES WITH INCREASE THE NUMBER OF PEPTIDE BONDS rumber of peptide bonds
Compound
Per cent of the fluorescence of ,the free;:;” neutral
Tyrosine
0
100
Gly-Tyr
1
Tyr-Gly Gly-Tyr-glycinamide
I 3
35 33 17
Tryptophan Gly-Try
0 1
100 28
TABLE QUANTUM PROTEINS
phosphate ions on the fluoresphenol. Indole + HPO4, pH f H2P04, pH 5.0 - - - -; pH 8.5 - - - - -; Phenol -k
H2P04-, pH 5.0 ........
Insulin Ribonuclease Pancreatic trypsin inhibitor Zein a Measured b Measured
pH
VIII
EFFICIENCY OF FLGORESCENCE WHICH CONTAIN ONLY TYROSINB THE FLUORESCENT COMPONENT
Protein
PIG. 3. Effect of cence of indole and 8.5 ---; Indole Phenol + HPO,-,
AND IN
in 70% (v/v) ethanol. in ethanol-water (50/5(l).
FOR AS
FLUORESCEKCE
OF INDOLE TABLE
EFFECT
OF CHANGES
IN
AND
43
PHENOL
IS
ON THE FLUORESCENCE OF PEPTIDES
IONIZATION
AND
Compound
OF TYROSINE
TRYPTOPHAN
PIOCW
n‘umber of atoms in the chain between the fluorescent ring and the ionic group
Huorescenceby the process
Per cent loss of
Gly-Tyr Leu-Tyr Tyr-Gly
-COO-COO--COO-
+ -COOH + -COOH + -COOH
2 2 5
62 62 32
Gly-Try Tryptophan My-Try
-COO-XH2 m-SH,
+ -COOH ---) --N+Ha --) -N+H:s
2 2 5
GI 56 40
length on the fluorescence of the tyrosyl residue is shown in Table VII. The peptide Gly-Tyr-glycinamide is probably representative of the effect of an extended peptide chain. The decrease in quantum efficiency by the attachment of the peptide chain (from 0.21 for tyrosine to 0.035 for GlyTyr-glycinamide) is sufficient to account in large measure for the low fluorescence of proteins which contain only tyrosine as the fluorescent amino acid (Table VIII). The average fluorescence of the four tyrosyl residues of insulin (62 = 0.037) is quite close to that for Gly-Tyr-glycinamide (& = 0.035). However, ribonuclease and pancreatic trypsin inhibitor show lower quantum efficiencies than would be expected from the electronegativity of the peptide chain alone. Other structural features that further lower the fluorescence of tyrosyl residues in these proteins will be described in later papers. (The quantum efficiency of zein is not comparable with the above compounds since the value was determined in an alcoholic solution for reasons of solubility.) Evidence for the effect of an extended peptide chain on the fluorescence of the tryptophyl residue is less satisfactory because of the limited number of peptides available for study. The change for glycyltryptophan in Table VII is similar t’o that for glycyltyrosine, and the effects of other substituents in Tables IV and VI lead to about the same loss of fluorescence for both tyrosine and tryptophan. Therefore, it is safe to say that the polypeptide chain will contribute substantially to the observed
(3) low fluorescence of tryptophyl residues in a number of proteins. The principal electronegative groups of amino acid side chains are the carboxyl groups of aspartic and glutamic acids and the positively charged groups of arginine, lysine, and histidine. In addition, terminal carboxyl and amino groups must be considered if the fluorescent amino acid is terminaLg However, the effectiveness of all these groups diminish rapidly with distance from the fluorescent ring as shown in Table IX. Even if an aspartyl residue were immediately adjacent to a tyrosyl residue on a peptide chain, the carboxyl group of the side chain would be separated from the fluorescent ring by a chain of six atoms. The inductive effect would be very small for the -COO- form, and even the transformation --COW + -COOH would be expected to decrease the fluorescence of the neighboring tyrosyl residue by less than 30% (by analogy with Tyr-Gly in Table IX). An effect of about the same magnitude might be expected for the posit.ively charged imidazolium ion of an adjacent histidyl residue. Even smaller effects would be cxpetted if tht: adjacent amino acid were glutamic acid, arginine, or lysine because of the further separation of t,he ionic charge y An amino-terminal tryptophyl group or a carboxyl-terminal trpptophyl or tyrosyl group would he expected to show marked changes in fluorescence with change of pH. Such changes should be similar to those in Fig. 2 for change in ionization of either t.he free carhoxyl or amino group.
44
COWGILL
from the fluorescent ring. Field effects that arise from the electrostatic charges of these ionic groups are more difficult to assess. Weber (10) has measured the quantum efficiency of fluorescence of tyrosyl residues in copolymers of tyrosine (4 %) and glutamic acid (96 5%). The quantum efficiency was very low (Q = 0.02) at neutral pH where the glutamyl side chains were in the -COOform. The quantum yield rose to Q = 0.13 at pH 2 where the glutamyl residues were in the -COOH form. These shifts in value of Q are in the direction opposite to that expected if pronounced inductive effects by the -COOH group occurred. Weber proposed that the -COOform of the glutamyl side chains quenched by the formation of a transit,ory phenolate ion. Such a proposal is certainly in accordance with Weber’s observations but would not account for the high fluorescence (Q = 0.13) in the presence of the polypeptide chain. An alternative proposal that might be considered is based on the change in shape and hydration of the copolymer. It must be assumed that a polymer which is 96 % glutamic acid and with the glutamyl residues in the ionic form must be in an expanded state and strongly hydrated. TJpon neutralization of these carboxyl ions by decrease in pH, the polymer should fold upon itself, and tyrosyl residues on the interior would be in a more hydrophobir environment. The fluorescence of the phenol ring is ronsiderably increased in such an environment (3).3 This explanation would be in harmony
with the observations of Weber and also would account, for the high quantum efficiency of fluorescence at low pH. Certaintly it appears likely that amino acid residues wit’h charged groups may affect the fluorescence of proteins in two ways. One is an inductive effect which is weak. The second is by electrostatic field effects which may be strong if a number of such ionic groups are concentrated in the vicinity of the fluorescent ring. ACKNOWLEDGMENT The valuable Elizabeth Moritz
technical is gratefully
assistance of acknowledged.
Mrs.
REFERENCES 1. SHORE, V. G., AND PARDEE, A. B., Arch. Biothem. Biophys. 60, 100 (1956). 2. TEALE, F. W. J., AND WEBER, G., Biochem. J 66, 476 (1957). 3. TFALE, F. W. J., Biochem. J. 76, 381 (1960). 4. DTJGGAN, ID. E., BOWMAN, R. L., BRODIE, B. B., AND UDENFRIEND, S., Arch. Biochem. Biophys. 66, 1 (1957). 5. WHITE, A., Biochem. J. 71, 217 (1959). G. PARKER, C. A., AND REES, W. T., ilnalyst 86, 587 (1960). and Phos7. PRINGSHEIM, P., “Fluorescence phorescence.” Interscience Publ., New York, 1949. 8. ENGSTROM, It. W., J. Opt. Sot. Am. 37, 420 (1947). 9. COHN, E. J., AND EDSALL, J. T., “Proteins, Amino Acids and Peptides.” Reinhold Pub]. Co., New York, 1943. 10. ROSENHECK, K., AND WEBER, G., Biochem. J. 79,29P (1961).
OF
BIOCHEMISTRY
Fluorescence
AND
BIOPHYSICS
and the Structure
on the Fluorescence
of Proteins.
of lndole
ROBERT From the Department
36-44 (1963)
I@,
of Biochemistry,
and
I. Effects of Substituents Phenol
W. COWGILL’, University Colorado
Received
of Colorado
August
Compounds
* School of Medicine,
Denver,
3, 1962
The quantum efficiency of fluorescence was measured for simple derivatives of indole and phenol and for tryptophyl and tyrosyl residues in peptides and proteins. Fluorescence of these compounds decreased as the electronegativity of substituents increased. In proteins, one strongly electronegative structure is the peptide bond. It is proposed that the observed, low fluorescence of tryptophyl and tyrosyl residues in proteins is attributable in large measure to the presence of this bond. INTRODUCTION
of tyrosine and tryptophan and ascribed losses in fluorescence to quenching by hydrogen ions, hydroxyl ions, and ionization of the phenolic hydroxyl group. The present study clearly reveals the high sensitivity of the fluorescence of both the indole and phenol rings to the electronegativity of substituents. In agreement with the earlier observations of Duggan et al. (4), increased electronegativity of substituents results in decreased fluorescence, and most of the observations of White (5) are explicable on this basis. One of the most powerful electronegative structures in proteins is the peptide bond, and suppression of fluorescence by the peptide chain is sufficient to account for the low fluorescence of some proteins.
The indole and phenol rings of tryptophan and tyrosine are fluorescent in proteins (1, 2). However, the quantum efficiency of fluorescence per tryptophyl or tyrosyl residue is not the same for all proteins (3) and is not even the same for different tryptophyl or tyrosyl residues in the protein molecule.3 This laboratory is now seeking the causes of these differences. The guiding hypothesis will be that the differences arise from variations in the local environment of the residues within or on the surface of the protein molecule, and the present paper is concerned with the inductive effects of groups near the aromatic ring. Duggan et al. (4) observed a loss of fluorescence upon acetylation of phenols and concluded that the fluorescence of the benzene ring depended upon the presence of a substituent (hydroxyl or amino group) able to donate electrons to the ring. White (5) studied the effect of pH on the fluorescence of peptides and other derivatives
MATERIALS
AND
METHODS
CHEMICALS Compounds for this study were obtained from sources indicated in Tables I and II. Identity and purity of the simpler compounds were established by absorption spectra, by fluorescence excitation and emission spectra, and by paper chromatography. Purification by distillation or crystallization was done when necessary. Identity and purity of peptides and other derivatives of tyrosine and tryptophan were established by the above tests as well as by hydrolysis to the amino acids. The latter were identified by paper chromatography. Insulin
1 Present address, Department of Biochemistry, Bowman Gray School of Medicine, Winston Salem, North Carolina. 2 This research was done during the tenure of a Research Career Development Award of The National Institutes of Health. 3 Unpublished work of this laboratory. 36
FLUORESCENCE
OF INDOLE
(tryst. Zn insulin, lot Xo. 7191Oti of Eli Lilly & Co.), pancreatic ribonuclease (chromatographitally pure, Type III, lot No. R22B-70 of Sigma trypsin inhibitor Chemical Co.), pancreatic (crystalline, lot, No. 1037 of Mann Research Laboratory), and zein (lot No. B2901 of Mann Research Laboratory) were used without purifical,ioll. ~~EASUREMENT
OF I~U~IWSCEIYCE
Fluorescence was measured in an AmincoBowman spectrophotofluorometer fitted with a 150-w. xenon lamp, slit. xsrangement I?r’o.3 and a Corning filter CS O-54 The filter removed scatter below 310 mp. Excitation and emission monochromators were calibrated with a mercury lamp. Fluorescence emission spectra were recorded on a chart recorder, and areas under the emission curves were determined by planimetry. Over the period of these measurements, the intensity of the xenon lamp varied because of variations in line voltage and the increasing age of the lamp. Since a comparison of quantum efficiency of fluorescence is valid only for constant light intensity, a correction was made in all measurements of fluorescence. The fluorescence of a reference solution (i nlg. tyramine. HCl/500 ml. water) was read at the same time as the measurement of fluorescence of a sample, and all readings were adjusted to a constant relative emission (R.E. = 1.98) for the t)-ramine reference solution. Fluorescence was measured in a concentration range t,hat ensured a linear relationship between fluorescence emission and concentration of the indole (lo-jlo* .&f) or phenol (10-4-10-” h1) compound. CALCULATION OF &IYAKTUM EFFICIBXCP 0~ FLUORESCXNCE Quant’urn efficiency of fluorescence was calculated by the method of Parker and Rees (6). This is a comparative method in which it is assumed that for a given spectrophotofluorometer the integrated area (A) under the fluorescence emission curve is proportional to the tot,al intensity of fluorescent light (F) emitted by the solution. The latter value (F) in turn is proportional to the where lo = intensity of excit.. product Za.Q.E.c.d, ing light, Q = quantum efficiency of fluorescence,4 E = molecular extinction coefficient, c = concent,ration, and d = optical path length. For comparison of fluorescence in t,he same apparatus and 4 The symbol Q is employed for quantum efficiency of fluorescence rather t,han t.he symbol + employed by Parker and Rees. The latter symbol has been employed for some time to denote energy yield of fluorescence [see Ref. (7), p. 3131.
8T
AND PHENOL
at the same intensity of excitation light, the ratio of fluorescence intensities is given by the equation:
F,
-z-z s;
A, A:
Q,.E,.c,.dl Qc.E2.cr.d?
= ($)(E$)
(l)
or
(2) Values for optical density at the wavelength of excitation and of the area under the observed emission spectral curve were determined for each compound. In order to avoid the necessity of COPrecting observed emission spectra for variation in intensity of the xenon lamp with wavelength of excitation, the exciting light was maintained constant at 280 rnr for all compounds. A second factor that may influence the emission spectrum is variation in t.he sensitivity of the photomultiplier tube to light of different wavelengths. Over the range of 300-400 rnp employed in this study, the RCA photomultiplier t,ube No. 11’ 28 is reported to vary in sensitivity of response by only 105, (8). This variation and any variat,ion in transmission of the emission monochromator have been neglected in measurement.s of areas under emission spectral curves.s. 6 Ratios of 3/O.D. for compounds bearing the indole ring and the phenol ring were compared with the values for tryptophan and tyrosine, respectively. Quantum efficiencies were calculated by Eq. (2) employing the reference values for Q of 0.20for t,ryptophan and 0.21 for tyrosine at neutral pH as reported by Teale and Weber (2). Values 6 Parker and Rees (6) have suggested that comparison of solutions may be based on intensity of fluorescence at the maximum of the emission curve once it has been established that, the ratio of area to spectral peak height is constant. This ratio is constant for hot.11 the tyrosine and tryptophan compounds of Tables IV and VI. However, the author prefers to rely on the area as measured by a planimeter since the ratio of area to peak height for tyrosine compounds in Table IV was found to vary from that for phenol compounds in Table III. Variations in this mtio have also been observed for f.ryptophan and tyrosine derivatives in other solvents and for tryptophyl and tyrosyl residues in different proteins. 6 In calculation of Q, t,he error from disregarding variations in the sensitivity of t,he photomult,iplier tube and in the transmission of the emission monochromator is estimated to be less t.han 5%. This error is small because t.he fluorescence spectral peaks are sharp and the fluorescence maxima are similar for compounds of both the indole and phenol t,ypes.
38
COWGILL TABLE
OPTICAL
ABSORPTION
AND
FLUORESCENCE
n-Tryptophan N-Ac-n-tryptophan rn-Tryptophan amide N-Ac-r-tryptophan methyl ester n-Tryptophan ethyl ester Glycyl-n-tryptophan Indole-3carboxylic acid Indole-3-acetic acid Indole
I
CHARACTERISTICS
OF INDOLE
AND
RELATED
COMPOUNDS”
278 280 278 -
6000 5900 5500 -
282 282 282 282
355 355 355 355
Mann Res. Lab. Lot No. A 5038 Mann Res. Lab. Lot No. A 4821 Sigma Chem. Co. Lot No. T6OB-061 HM Chem. Co. Lot No. 113-100-19
278 280 278 279 278
6000 5800 8400 7000 5606
282 282 282 282 282
355 355 360 365 355
Mann Res. Lab. Lot No. B 1978 Mann Res. Lab. Lot No. 1438 K & K Labs. Lot No. 31518 K & K Labs. Lot No. 20988 Fisher Sci. Co. Lot No. 710800
QX, and +, are respectively the optical absorption maximum in millimicrons and the molar extinction coefficient in aqueous solution at pH 7. Neither acidity not basicity of the solution affected the spectra of the indole compounds markedly. Acidity of the solution did not affect the spectra of the A, and Em are the wavelength maxima in millimicrons of the fluorescence phenol compounds markedly. activation and fluorescence emission spectra, respectively. These latter values have not been corrected for idiosyncrasies of either the xenon lamp or the IP28 photocell of the spectrophotofluorometer. TABLE OPTICAL
ABSORPTION
AND
FLUORESCENCE
II
CHARACTERISTICS
OF PHENOL
AND
RELATED
COMPOUNDS~
Compound
m-Cresol m-OH benzyl alcohol Phenol m-OH benzoic acid m-OH benzaldehyde
270 272 268 287 311
1750 1600 1500 2100 2700
279 280 278 295 -
320 320 320 413 -
K & K Labs. K & K Labs. Mallinckrodt K & K Labs. K & K Labs.
nn-Tyrosine n-Tyrosylglycine Glycyl-n-tyrosine Tyramine Glycyl-n-tyrosylglycinamide Leucyl-n-tyrosine
274 274 274 274 274
1520 1460 1280 1480 1480
285 285 285 285 285
320 320 320 320 320
Nutritional Biochem Mann Research Labs. Mann Research Labs. Mann Research Labs. Mann Research Labs.
274
1360
285
320
Mann
(1See footnote
to Table
calculated in this fashion reported in the literature Table VIII. RESULTS
AND
Research
Lot Lot USP Lot Lot
No. 10429 No. 31850 Cryst ., “Gilt No. 15657L No. 3072OL Co. Lot Lot Lot Lot
Lot No. No. No. No.
Label”
No. 8060 C 1054 D 4280 CA 2841 C 1151
Labs. Lot No. E 2273
I. compare well with those (3, 5), for example, see DISCUSSION
The compounds for this study are listed in Tables I and II with values for optical absorption maxima, molar extinction coefficients, fluorescence excitation maxima, and emission maxima. It may be observed that these values are similar for the different compounds of both the indole and the phenol series. Therefore, the nature of the
substituent has no appreciable effect on the excitation or emission maxima.7 The change which does occur with change of structure is in the intensity of fluorescence, that is, the quantum efficiency (Q) of fluorescence. The term (Q) may be defined as the ratio of the number of quanta of light emitted as fluorescence/number of quanta of light absorbed by the fluorescent substance. Values for Q have been determined by the com7 One exception in this benzoic acid of Table II.
series
is nL-hydroxy-
FLUORESCENCE TABLE QUANTUM
III
I~FFICIENCY (CJ) OF FIXORESCENCE META-SUBSTITTITED PHENOLS
-CHa -CH,OH
-H -coo-COOH
-CHO
OF INI~OLE
FOR
0.25 0.25 0.21 0.05 0.01
< 0.01
parative method described in the preceding section. COMPOUNDS THAT CONTAIN PHENOL RIKG
THE
The marked effects on the quantum efficiency of fluorescence by substituents introduced directly on the phenolic ring are shown in Table III. The loss of fluorescence is in the order to be expected on the premise that substituents of increasing electronegativity (that is, substituents that have an increasing ability to pull electrons from the ring) lead to decreasing fluorescence. Derivatives with hydroxymethyl or carboxaldehyde substituents at the ortho or para positions gave the same Q values as did the corresponding meta derivatives in Table III. So the position of the substituent on the benzene ring had no influence on the fluorescence of these compounds.R The phenolic rings of tyrosine and peptides containing tyrosine also show loss of fluorescence with the introduction of increasingly electronegative groups. The principal electronegative groups in these compounds have been underscored in Table IV. In these compounds, the inductive effect is attenuated by t.he intervening side chain so that the loss of fluorescence is not as marked as for compounds in Table III. There are 8 This conclusion does not apply to isomers in which the absorption spectrum is different for the various ring positions of substituents; for example, the hydroxpbenzoie acids.
AND
39
I’HENOI,
indications in Table IV that factors other than electronegativity influence the fluorescence of tyrosine in peptides, for example, compare leucyltyrosine (Q = 0.103) and glycyltyrosine (Q = 0.070). The influence of various amino acid side chains on the quantum efficiency of fluorescence will be considered in a second paper. COMPOUNDS THAT COSTAIN INJDOLX Rrsc,
THE
Efiects of electronegative substituents on the quantum efficiency of fluorescence of the indole ring are shown in Table V. The loss of fluorescence is greater for a -OH group than for the -COOform as would be expected from the greater electron-attracting power of the former group. Also, the attenuat,ion of the effect, by the interposition of even one methylene group between t,he electronegative substituent and the ring is clear. The effect of an electronegative group is further attenuated in peptides and other derivatives of tryptophan listed in Table VI. For example, compare indole-COO(Q = 0.24), indole-CHzCOO- (Q = 0.38), and indole-CHzCH(XH,)-COO(Q = 0.51). A carboxyl group in the solution but not a part of the fluorescent molecule has no comparable action on the fluorescence of either the indole or phenol ring. Both acetic acid and acetate ions weakly quench the fluorescence of tyrosine. The data conform with the Stern-Volmer equation for fluorescence quenching, and concentrations for half-quenching are 0.8 M acetic acid and 0.15 11f sodium acetate. Similarly, 0.8 Al acetic acid is required t,o half-quench tryptophan, but sodium acetate shows no quenching of tryptophan even at 0.4 A/. Quenching of tyrosine by other carboxylic acids in the same concentration range (0.1-0.8 M) has been reported by Teale (3). Certainly this quenching effect requires concentrations of the carboxylic acid several orders of magnitude higher than the concentration of compounds in Tables III-VI. Also, there is other evidence that such quenching is quite unrelated to the inductive effect of carboxyl groups attached to the fluorescent molecule. For example,
40
COWGILL TABLE QUANTUM EFFICIENCY
OF
IV
FLUORESCENCE FOR TYROSINE HO-’
0
PEPTIDES
AND
CONTAININGTYROSINE
’ -CHzCH-RI
-
Compound
RX
R2
RI
Tyrosine
-coo-
Tyramine
-H
HsN+H&+-
Leu-Tyr
--coo-
H~N+-cH--C~--?~TH-
-CO--SH-CH,-COO-coo-
Tyr-Gly Gly-Tyr
I Leu
0.103
-
H.N+-
0.074
HaN+-CHs-CO-NH-
0.070
HaN+-
0.056 0.050
-CO--NH-CH?-a-NH2 -CZIH
H3N+-CHy-CO-NH-
0.027
Gly-Tyr-glycinamide Gly-Tyr
V
QUANTUMEFFICIENCYOF FLTJORESCENCE FOR INDOLE COMPOUNDS
-H -CH&OO-coo-CH&OOH -COOK
0.185
-
-COOH AX--NH-CH,-COOH
TABLE
0.21
HsN+Hd+-CHz-CO-NH-
Tyr-Gly
Tyrosine
Q
0.40 0.38 0.24 0.16 0.08
the acetate ion is more effective than acetic acid for quenching tyrosine while the -COOH group is more effective than the -COOionic form for decreasing the fluorescence of the compounds in Tables III-VI. Also, the acetate ion has no quenching effect on tryptophan while the attached -COOgroup has a comparable effect on both the indole and phenol compounds of Tables III-VI. EFFECTS OF pH AND BUFFERS ON FLUORESCENCE Fluorescence was measured in aqueous solutions that were adjusted to insure the
-
0.035
presence of the desired ionic species of the fluorescent compound. For this purpose, measurements were made in 0.01 N HCl, 0.05 M Tris buffer of pH 7.5 for the phenol compounds, and in 0.005 M phosphate buffer of pH 6.0 or 10.5 for the indole compounds. White (5) has reported quenching of the fluorescence of tyrosine and tryptophan derivatives in hydrochloric acid. In the present studies, no marked quenching effect of WC1 on the fluorescence of phenol compounds was noted although the fluorescence of indole compounds is more sensitive to this acid (Fig. 1). It would appear that some of the changes in fluorescence with change in pH, which White ascribed to quenching by hydrogen ions, were actually due to changes in ionic species of the compound. Figure 2 shows the effect of pH on the quantum efficiency of fluorescence of tyrosine and tryptophan. These curves are similar in shape and points of inflection to curves given by White. 1% will be observed in Fig. 2 that the quantum efficiency of fluorescence is constant for both tyrosine and tryptophan in the region of pH 4-8. In more acidic solutions the Q values fall for both amino acids, and the pH at the midpoints of these decreases
FLUORESCENCE
OF INDOLE
ANI)
PHENOL
41
TABLE VI QUANTUM
OF FI,.L;ORESCENCE FOR
EFFICIXNCY
Compound
0.51
-coo-
CH,-CO-NH-
0.28
-CJ
H&-
0.28
-coo-
H$+-
0.20
-co- NH? -COOH -
H 2 U+-
0.20
CH&SO-NHHa?U’+-
0.128 0.080
WAC-tryptophan nmidc
Tryptophnn amide
K-Ac-tryptophan
Q
HZN--
-coo-
Tryptophan
RZ
RI
Tryptophnn Tryptophnn
NH?
0.085
F-AC Me ester of tryptophan
-COOH -COOCH3 -
EC ester of tryptophan
-COOC2Hs -
CH,-CO-NHH,N-
El ester of tryptophan
-COOCrHj -
H3?1;+-
0.032
Gly-Try
-coo-
0.095
Gly-Try
-coo-
H?S-CHT-CO-SHHay+-CHr-CO-NH-
0.057
Gly-Tr)
-coo1
&S+-CHZ-CO-XH-
0.022
TrZ’ptopllall
:. E To
TRYPTOPHAN, ITS PEPTIDES AND OTHERI)ERIvATIvEs
--...-..-.
-..._.-
.-.-.-.-.a
.-.-._.
0.076
-.-.m.
3..\ -,:““-,--
E
--------..----------_,,__
-*---mm.
..\
.t
.
A., .
.
. y-1.
0 I-
.---_ ---__
.
FIG. 1. Effect of hydrochloric acid on the fluorescence of peptides of tyrosine and tryptophan. Tyrosylglycine (1 X 1OP M) ----; Leucyltyrosine (1 X IOV J!f) - - - - -; GlJwyltyrosine (1 X lo-” ,!I) - - - - - - -; J,eucyltryptophan (I X lo-” Jf) - - -- -; Blycylt.ryptophan (1 x lo-j!ll) . . .
correspond to the dissociation constants (9) of the carboxyl groups (for tyrosine, pK1 = 2.20; for tryptophan, pK1 = 2.38). Similarly, changes in the alkaline region of pH 8-11 can be explained by changes in ionization. As mentioned by White, the fall in fluo-
Frc:. 2. Effect of pH on the quantum efficiency of fluorescence. Tyrosine -n-n--; Tryptophan -O-O-; Methyl ester of S-acetyltryptophan --O--O-. (No other hufer was added.)
rcscence of tyrosine occurs with a midpoint at pH 10.0 which compares well with its pKa = 10.06 (9) for ionization of the hydroxyl group and confirms previous observations that the ionized phenolic ring is nonfluorescent for tyrosine and its derivatives. The rise in fluorescence of tryptophan with a midpoint at pH 9.5 corresponds to
42
COWGILL
the dissociation of a proton from the amino group with p& = 9.39 (9). (Conversion of a -NH,+ group to a less electronegative --NH2 group would be expected to increase the fluorescence of the indole ring.) Beyond pH 11, tryptophan also loses fluorescence ; and this is presumably due to the quenching action of the hydroxyl ion as described by White. The proposal that changes in fluorescence in the regions of pH 13 and pH 8-11 are due to changes in ionic structure, is substantiated by the curve for the methyl ester of acetyltryptophan in Fig. 2. This ester displays neither the fall in fluorescence at low pH nor the rise in the region of pH 8-11. These differences from the curve for tryptophan are to be expected since the carboxyl group is esterified and the amino group is acetylated so that the molecule does not undergo change of structure with change of pH. Figure 2 should serve to emphasize the fact that values for & in Tables III-VI are approximations for those structural forms which exist only at high acidity or alkalinity. This is so because conditions chosen for measurement in 0.01 N HCl or in buffer at pH 10.5 do not necessarily correspond in all cases to minima or maxima of curves similar to those in Fig. 2. Tris buffer at 0.05 M and pH 7.5 showed no quenching effects with either indole or phenol compounds. This buffer was used in preference to phosphate for the phenol
compounds because fluorescence of the latter was strongly quenched by phosphate. As may be observed in Pig. 3, the HP04-ionic species is more effective than H,PO,for quenching fluorescence of phenol. The fluorescence of indole is weakly quenched by phosphate, but the more effective ionic species is H2P04- rather than HPO,-. RELATIONSHIP BETWEEN THESE OBSERVATIONS .4m THE FL~ORERCEY~CE OF TRYPTOPHAK AND TYROSINE IN
PROTEINS
The bearing that observations made in this paper have on fluorescence of proteins must be considered from two aspects. One is the effect of the peptide chain itself; the second is the effect of electronegative amino acid residues along the pept.ide chain. The effect of increasing peptide chain TABLE
VII
DECREASE IN FLUORESCENCE OF TYROSYL TRYPTOPHYL RESIDUES WITH INCREASE THE NUMBER OF PEPTIDE BONDS rumber of peptide bonds
Compound
Per cent of the fluorescence of ,the free;:;” neutral
Tyrosine
0
100
Gly-Tyr
1
Tyr-Gly Gly-Tyr-glycinamide
I 3
35 33 17
Tryptophan Gly-Try
0 1
100 28
TABLE QUANTUM PROTEINS
phosphate ions on the fluoresphenol. Indole + HPO4, pH f H2P04, pH 5.0 - - - -; pH 8.5 - - - - -; Phenol -k
H2P04-, pH 5.0 ........
Insulin Ribonuclease Pancreatic trypsin inhibitor Zein a Measured b Measured
pH
VIII
EFFICIENCY OF FLGORESCENCE WHICH CONTAIN ONLY TYROSINB THE FLUORESCENT COMPONENT
Protein
PIG. 3. Effect of cence of indole and 8.5 ---; Indole Phenol + HPO,-,
AND IN
in 70% (v/v) ethanol. in ethanol-water (50/5(l).
FOR AS
FLUORESCEKCE
OF INDOLE TABLE
EFFECT
OF CHANGES
IN
AND
43
PHENOL
IS
ON THE FLUORESCENCE OF PEPTIDES
IONIZATION
AND
Compound
OF TYROSINE
TRYPTOPHAN
PIOCW
n‘umber of atoms in the chain between the fluorescent ring and the ionic group
Huorescenceby the process
Per cent loss of
Gly-Tyr Leu-Tyr Tyr-Gly
-COO-COO--COO-
+ -COOH + -COOH + -COOH
2 2 5
62 62 32
Gly-Try Tryptophan My-Try
-COO-XH2 m-SH,
+ -COOH ---) --N+Ha --) -N+H:s
2 2 5
GI 56 40
length on the fluorescence of the tyrosyl residue is shown in Table VII. The peptide Gly-Tyr-glycinamide is probably representative of the effect of an extended peptide chain. The decrease in quantum efficiency by the attachment of the peptide chain (from 0.21 for tyrosine to 0.035 for GlyTyr-glycinamide) is sufficient to account in large measure for the low fluorescence of proteins which contain only tyrosine as the fluorescent amino acid (Table VIII). The average fluorescence of the four tyrosyl residues of insulin (62 = 0.037) is quite close to that for Gly-Tyr-glycinamide (& = 0.035). However, ribonuclease and pancreatic trypsin inhibitor show lower quantum efficiencies than would be expected from the electronegativity of the peptide chain alone. Other structural features that further lower the fluorescence of tyrosyl residues in these proteins will be described in later papers. (The quantum efficiency of zein is not comparable with the above compounds since the value was determined in an alcoholic solution for reasons of solubility.) Evidence for the effect of an extended peptide chain on the fluorescence of the tryptophyl residue is less satisfactory because of the limited number of peptides available for study. The change for glycyltryptophan in Table VII is similar t’o that for glycyltyrosine, and the effects of other substituents in Tables IV and VI lead to about the same loss of fluorescence for both tyrosine and tryptophan. Therefore, it is safe to say that the polypeptide chain will contribute substantially to the observed
(3) low fluorescence of tryptophyl residues in a number of proteins. The principal electronegative groups of amino acid side chains are the carboxyl groups of aspartic and glutamic acids and the positively charged groups of arginine, lysine, and histidine. In addition, terminal carboxyl and amino groups must be considered if the fluorescent amino acid is terminaLg However, the effectiveness of all these groups diminish rapidly with distance from the fluorescent ring as shown in Table IX. Even if an aspartyl residue were immediately adjacent to a tyrosyl residue on a peptide chain, the carboxyl group of the side chain would be separated from the fluorescent ring by a chain of six atoms. The inductive effect would be very small for the -COO- form, and even the transformation --COW + -COOH would be expected to decrease the fluorescence of the neighboring tyrosyl residue by less than 30% (by analogy with Tyr-Gly in Table IX). An effect of about the same magnitude might be expected for the posit.ively charged imidazolium ion of an adjacent histidyl residue. Even smaller effects would be cxpetted if tht: adjacent amino acid were glutamic acid, arginine, or lysine because of the further separation of t,he ionic charge y An amino-terminal tryptophyl group or a carboxyl-terminal trpptophyl or tyrosyl group would he expected to show marked changes in fluorescence with change of pH. Such changes should be similar to those in Fig. 2 for change in ionization of either t.he free carhoxyl or amino group.
44
COWGILL
from the fluorescent ring. Field effects that arise from the electrostatic charges of these ionic groups are more difficult to assess. Weber (10) has measured the quantum efficiency of fluorescence of tyrosyl residues in copolymers of tyrosine (4 %) and glutamic acid (96 5%). The quantum efficiency was very low (Q = 0.02) at neutral pH where the glutamyl side chains were in the -COOform. The quantum yield rose to Q = 0.13 at pH 2 where the glutamyl residues were in the -COOH form. These shifts in value of Q are in the direction opposite to that expected if pronounced inductive effects by the -COOH group occurred. Weber proposed that the -COOform of the glutamyl side chains quenched by the formation of a transit,ory phenolate ion. Such a proposal is certainly in accordance with Weber’s observations but would not account for the high fluorescence (Q = 0.13) in the presence of the polypeptide chain. An alternative proposal that might be considered is based on the change in shape and hydration of the copolymer. It must be assumed that a polymer which is 96 % glutamic acid and with the glutamyl residues in the ionic form must be in an expanded state and strongly hydrated. TJpon neutralization of these carboxyl ions by decrease in pH, the polymer should fold upon itself, and tyrosyl residues on the interior would be in a more hydrophobir environment. The fluorescence of the phenol ring is ronsiderably increased in such an environment (3).3 This explanation would be in harmony
with the observations of Weber and also would account, for the high quantum efficiency of fluorescence at low pH. Certaintly it appears likely that amino acid residues wit’h charged groups may affect the fluorescence of proteins in two ways. One is an inductive effect which is weak. The second is by electrostatic field effects which may be strong if a number of such ionic groups are concentrated in the vicinity of the fluorescent ring. ACKNOWLEDGMENT The valuable Elizabeth Moritz
technical is gratefully
assistance of acknowledged.
Mrs.
REFERENCES 1. SHORE, V. G., AND PARDEE, A. B., Arch. Biothem. Biophys. 60, 100 (1956). 2. TEALE, F. W. J., AND WEBER, G., Biochem. J 66, 476 (1957). 3. TFALE, F. W. J., Biochem. J. 76, 381 (1960). 4. DTJGGAN, ID. E., BOWMAN, R. L., BRODIE, B. B., AND UDENFRIEND, S., Arch. Biochem. Biophys. 66, 1 (1957). 5. WHITE, A., Biochem. J. 71, 217 (1959). G. PARKER, C. A., AND REES, W. T., ilnalyst 86, 587 (1960). and Phos7. PRINGSHEIM, P., “Fluorescence phorescence.” Interscience Publ., New York, 1949. 8. ENGSTROM, It. W., J. Opt. Sot. Am. 37, 420 (1947). 9. COHN, E. J., AND EDSALL, J. T., “Proteins, Amino Acids and Peptides.” Reinhold Pub]. Co., New York, 1943. 10. ROSENHECK, K., AND WEBER, G., Biochem. J. 79,29P (1961).