The properties of some phenylalanyl peptides in ethylene glycol-aqueous buffer solvent

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The

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Properties

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of Some

Phenylalanyl

Glycol-Aqueous IRA Laboratory

of Ph!ysical

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Peptides

Buffer ROBERT

AND

Naval

Medical

in Ethylene

Solvent’ F. STEIKER

Research

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20014 Received

October

8, 1968;

accepted

February

1, 1969

The behavior of several phenylalanyl dipeptides in a mixed ethylene glycolaqueous buffer solvent has been studied by spectroscopic techniqlles, and definite evidence for a concentration-dependent self-interaction has been obtained for these systems. Of particular interest is the observation of gel formation, suggestive of linear aggregation, for N-l~enzyloxycarbonyl-~-phenylalanyl-~-p~~e~~ylalanine and for L-methiollyl-L-phenylalanine under suitable conditions of concentration, temperature, and mixed solvent composition. This evidence of aggregation is in accord with t,heoretical consideratiotls of the structrlre of biopolymers.

form of linear aggregation. In this paper WC present physical data upon the solution properties of this and related systems.

Research into the lowtempcrature luminescence of proteins, pcptides, and related model compounds (l-6) has been aided by the development of organic solvents and organic solvent-water mixtures which form transparent glasses at liquid nitrogen temperatures. Accordingly, detailed interpretations of the data obtained will ultimately depend on an understanding of the physicochemical properties of these compounds in such solvent systems. To facilitate interpretation of the lo\v-temperature luminescence of phenylalanyl peptides, n-e have initiated studies of their properties in ethylene glycolwater mixtures, the most popular solvents for such investigations. In the course of these background studies, we have observed tha,t N-bensyloxycarbony1 - L - phenylalanyl -L - phenylalanine (ZPhe-Phe) forms a thixotropic gel in waterethylene glycol mixtures under restricted conditions of concentration, temperature, and solvent composition. Gelation may usually be regarded as an indication of some

EXPERIMENTAL

PROCEDURE

Materials. I,-phenylalanine (Phe) was a Sigma Grade reagent of the Sigma Chemical Co. (St. Louis, h40.). Two samples of iV-benzyloxycarbonyl-L-phenylalanyl-L-phenylalanine (Z-PhePhe) were used. One of these was obtained as a Grade I preparation from the Cycle Chemical Corp. (Los Angeles, Calif.); the other was prepared by Dr. M. Wilcheck of the Weizmann Institute, Rehovoth, Israel, and was obtained through the court’esy of Dr. H. Edelhoch of the National Institutes of Health. The two preparations gave equivalent results. Samples of L-phenylalanyl glycine (Phe-Gly) from Cycle (Grade I) and Miles Laboratories (Elkhart, Ind.) also gave equivalent results. I,leucyl-L-phenylalallirle (Leu-Phe) and L-methionyl-I,-phenylalanil~e (Met-Phe) were Grade I products of Cycle. L-phenylalanyl-L-leucine (PheLeu), L-phenylalanyl-L-alanine (Phe-Ala) and Lphenylalanyl-r-phenylalanine (Phe-Phe) were &liles prodltcts. The above compounds were used as received. The solvents employed consisted of ethylene glycol and aqueolls buffer in varying proportions. The aqueorts solvent, was 0.05 M in potassium phosphate bluffer, pH 7.0, unless otherwise noted. Chromato-quality Reagent ethylene glycol

1 Research Task No. ~111005.06.0005. The opinions in this paper are those of the authors and do not necessarily reflect the views of the Navy Department or the naval service at-large. 2 NAS-NRC Research Associate. 263

264

WEINRYB

AND

(Matheson, Coleman, and Bell, East Rutherford, N. J.) was used for some of the luminescence measurements. Other reagcllts were Fisher Scientific Certified Reagent materials. Concentration-difference spectroscopy. A Cary Model 14 recording spectrophotometer was used to obtain concentration-difference spectra in the ultraviolet wavelength region. The (ungelled) solution of higher concentration was placed in a short pathlength quartz cuvette kindly provided by Dr. Gary Felsenfeld of the National Institutes of Health; its pathlength was determined to be 1.009 mm by spectral comparison with a 1.000.cm cuvette. Ten-fold differences in concentration were studied. The dilute solution was placed in a standard l.OOO-cm cuvette. Solvent baselines were obtained in each case over the entire wavelength range. The wavelength scan was made with the slit width automatically controlled and was discontinued when a slit width of 2.9 mm was reached. Dynode tap setting 3 was generally used. Since the total absorbances ranged as high as 2.5, the possibility existed of artifacts arising from stray light. These were checked for by observation of the spectrum obtained when l-cm cuvettes filled with the dilute Z-Phe-Phe solution were placed in both the sample and reference compartments; no absorption above the baseline was observed. As a further check against the existence of artifacts, a difference spectrum was also obtained for which the concentrated solution was placed in a l.OOO-cm cuvette equipped with a silica spacer (Beckman Instruments), which reduced the pathlength to 1.000 mm. The results for equivalent solutions were the same as for the arrangement described above. The problem of dilution errors is discussed in the Results section. Optical activity measurements. Circular dichroism (CD) spectra were obtained with a Cary Model 60 spectropolarimeter equipped with a circular dichroism accessory. We thank Dr. Harold Edelhoch and Mr. R. E. Lippoldt of the National Institutes of Health for providing access to this instrument. Baselines were determined with solvent in the identical cell used for the sample spectrum. Oneand ten-millimeter pathlength cells were used. The spectra are presented in terms of the recorded ellipticity (degrees), since they are intended essentially for comparison purposes. Direct comparisons between concentrated (ungelled) and approximately ten-fold dilute sample solutions are effected by normalization of the spectra, when necessary, to equivalent total sample absorbances. Ellipticity values for the given sample were reproducible to within &15% at wavelengths greater than 245 mp (where spectral amplitudes were small) and to within ~5% at wave-

STEINER lengths below abollt 245 mr. The wavelength location of spectral features could be det,ermined to f0.5 mp. Luminescence experiments. Fluorescence quenching studies at room temperature, determinations of total emission spectra at low temperatures, and measurements of phosphorescence lifetimes were carried out on an Aminco-Bowman spectrofluorometer equipped with a spectral compensation attachment which corrects emission spectra for wavelength variation in detector response. Experiments at liquid nitrogen temperatures were done utilizing an automatic liquid nit,rogen level controller. The equilibrium temperature was 91” K. Details of the experimental procedures followed have been given elsewhere (4,7). Measurements of fluorescence polarization were made using horizontally and vertically oriented Polacoat disks, which were supplied through the courtesy of Dr. Raymond Chen of the National Institutes of Health. Unpolarized incident light was used. A correction factor was applied for the polarizing effect of the monochromatizing system. This was determined using t.yrosine in water at 25” as a reference emitter of zero polarization. The polarization (P) was computed from the relationship : P

=

(I,

-

Ih)/(I,

where Ih and I, are the intensities and vertically polarized fluorescent spectively.

+

Ih),

of horizontally radiation,

re-

RESULTS

Gelation of Z-Phe-Phe

solutions. Gel forfor Z-Phe-Phe in pure ethylene glycol or aqueous buffer. The occurrence of gelation in mixtures of the two solvents was dependent upon the Z-Phe-Phe concentration, the temperature, and the solvent composition. Gelation was favored by low temperature, high Z-Phe-Phe concentration, and by increasingly high proportions of water (for volume fractions of water in the range o-0.5). Under conditions where the solubility limits of Z-Phe-Phe in the mixed solvent were approached, the gels were turbid. At lower Z-Phe-Phe concentrations, under otherwise equivalent conditions, water-clear gels were obtained. Figure 1 shows an example of each type. When examined visually by transmitted light some gels showed indications of gradients of refractive index. Melting of the gels to form fluid solutions

mation

was

not

observed

PROPERTIES

07

PHENYLALANYL

FIG. 1. The elt’cct of %-Phe-Phe concentration on the appearance of %-Phe-Phe gels. Left: Tllrhid gel; 11 mg/ml. Right: Virtually clear gel; 7.5 mg/ ml . Solvent : TT’ ‘I EC+. Note weights suspended by I the gels ill the inverted tllbes.

occurred in all cases at sufficiently high temperatures. The melting temperature was determined visually, the criterion being the ability of a small, glass-enclosed weight to move freely through the solution upon in version of the test tube. By this criterion the sol-gel transition was relatively sharp, attaining completion over a temperature range not exceeding l-2”. Table I shows the melting temperatures as a function of solvent composition for two Z-Phe-Phe concentrations. It is clear that the melting point increases sharply with increasing water content and with increasing Z-Phe-l’he concentration. The melting temperatures of Table I are for mixtures which had stood at’ 3” for 24 hr. Table I also includes the observed gelation temperatures for solutions which had been preheated for 20 min at 100” and then maintained at the given temperature for 5

PEPTIDES

%A5

min. The gelation temperatures obtained in this way were invariably lower than the melting temperatures, reflecting the .slon-ness of the gelation process. The amount of Z-Phe-Phe present in the gel phase is dependent upon the concentration of Z-Phe-Phc initially present. l:or example, solutions containing 13.4 and 7.7 mg/ml of Z-Phe-Phe (in 77’; ethylene glycol-23 ?i 0.05 M phosphate, pEI 7.0) both gelled after 24 hr at 3”. The gels were spun at 30,000 rpm for 2 hr in a Spinco Alode I, ultracentrifuge. The gel phases n-we thercby partially separated from the supcrnntant, solutions, being compressed to about ?$ of the total volume. The supernatant absorbawes at 25s rnp n-ere compared with the absorbantes of the initial solution prior to gclation. In this way it was found that the gel phase accounted for about 60 ‘; of the Z-Phe-Phe initially present in the more concentrated sample (15.4 mg/ml), but for only about .550 in the less concentrated (7.7 mgjml). Sigthe supernatnnt absorbancies nificantly, \verc similar for the two, as would be expccted if all Z-Phe-Phc in excess of n critical concentration were incorporated into the gel. The only other system for \\-hich gelation TABLE

I

EFFECT OF H~I,VEXT C~MPOSI,~I~N, CON~ENTRATIOS, AND TEMPERXNRE

%-Phe-Phe ox GEL

FORMATION

100/o

10

20 02/8

10

83/17

20 10 20

75/25

10

67133

20 10 20

<8 <8 <8 <8 <8 32 35.5 54 41 67.5


>42

a Samples warmed from starting temperature of 8” melted when held at stated temperature for 5 min. b Melted samples were cooled from 100” (20 min.) and gelled when held at stated temperature for 5 min.

266

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WEINRYB

AND

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STEINER

is not believed to associate over this concentration range, were constant, apart from scatter, over this wavelength range and were devoid of detail, with AA/A = 0.015-0.02. In this case, the entire difference spectrum arises from dilution error. L-Phe in PO, buffer (pH 7) and Z-Phe-Phe in 8 M urea or in 100% EG showed essentially no significant concentration-difference spectra. It was not feasible to examine Z-Phe-Phe in aqueous solution because of its limited solubility. Figure 2 shows a representative normalized Z-Phe-Phe difference spectrum in 80% ethylene glycol (1.5% vs. 0.15%). The normalized spectrum shows definite structure, with a large positive, or “red-shifted” peak at about 238 rnp and smaller positive

OF

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220

230

240

250

260

270

280

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WAVELENGTH Impi

FIG. 2. Concentration-difference spectra for Z-Phe-Phe, Phe-Phe, and Phe in 8O70 EG, and for Met-Phe in 80 and 50y0 EG, normalized to the absorbance of the reference (dilute) solution at each wavelength; A258.6 mp for the reference solutions were 2.11, 1.33, 2.00, 0.87, and 0.72, respectively. The spectra for Met-Phe have been displaced upward by 0.15 and the spectrum for Phe displaced downward by 0.05 for clarity of presentation.

was observed was Met-Phe, which formed turbid gels (>O.S % Met-Phe, 50 % EC). Concentration-difference spectroscopy. Since the gelation of Z-Phe-Phe is suggestive of some form of concentration-dependent intermolecular interaction, the technique of concentration-difference spectroscopy (8, 9) was employed as a further means of characterizing the process. The chief source of error in this type of measurement is random or systematic dilution error, so that the ratio of concentrations is not precisely equal to the inverse of the ratio of pathlength. If the difference spectrum is normalized with respect to the total absorbance at each wavelength, the component arising from dilution error should be constant and independent of wavelength. Normalized concentration-difference spectra of bovine serum albumin (0.1 M phosphate, pH 7; 2.5%) vs. 0.28%), which

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250 WAVELENGTH

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E‘IG. 3. Concentration-difference spectra for Phe-Ala, Phe-Leu, and Phe-Gly in 80% EG, normalized to the absorbance of the reference (dilute) solution at each wavelength. Azs,.: rnp for the reference solutions were 2.10, 1.86, and 2.50, respectively. The spectrum for Phe-Leu has been displaced downward by 0.10 and that for Phe-Gly by 0.20 for clarity of presentation.

PR.OPERTIES

OF

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250 WAVELENGTH

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FIG. 4. Circular dichroism spectra (23&380 rnk) of concentrated (-) and about ten-fold dilute (-----) solutions of Phe in 71% EG. Spectra normalized to equivalent total sample absorbantes. Az5a.s rnp = 0.845/mm for concentrated sample. Vertical lines indicate locations of corresponding absorption peaks for each solution.

PEPTIDES

267

Circular dichroism spectra. The optical activity of a chromophore in solution is knotvn to be highly sensitive to the details of its microenvironment,. In particular, small changes in the conformation of the chromophore-containing compound may give rise to discernible changes in optical activity of the chromophore. The examination of the CD of such compounds is especially informative due to the relative easeof assignment of CD bands to the corresponding absorption bands (10). The CD spectrum of a concentrated and a dilute Phe solution in 71% EG arc shown in Fig. 4. The jvavelength locations of the corresponding absorption peaks are included for each solution. Although the optical activity of the electronic transitions of Phe in the 245-270 rnp range is exceedingly \veak (10, 11) (the molar ellipticity, [e], at 258 rnp, -60 for these solutions), dichroic bands corresponding to five of the absorption bands can be distinguished in t,his region. Within

I i

peaks in the 250-270 rnp region. The strong positive peak at 238 rnp did not appear for any unblocked phenylalanyl peptides (including Phe-Phe) and thus probably arises from an environmental change of the beneyloxycarbonyl group itself. Phe in 80% ethylene glycol (Fig. 2) showed a negative (blue-shifted) peak at about 227 ml. In SO% ethylene glycol, three phenylalanyl dipeptides, Phe-Gly, Phe-Leu, and I’he-Ala, gave strongly negative difference spectra in the 250-270 rnp region, corresponding to a “blue-shift” of the primary phenylalanine band (Fig. 3). In all three dipeptides, the a-amino group is unblocked and phenylalanine is at the N-terminal end. In contrast, Leu-Phe, Met-Phe, and l’hePhe all gave positive difference spectra in ethylene glycol-water solutions (Fig. 2). It is of interest that Phe-Leu and Leu-Phe show qualitatively different spectra, indicating that a shift of phenylalanine to the C-terminal position alters the character of the interaction.

FIG. 5. Circular dichroism spectra (235-290 rnp) of concentrated (+-) and about ten-fold dilute (-----) solutions of Phe-Phe in 717, EG. Spectra normalized to equivalent total sample absorbances. A2s8.6 nqu = 0.700/mm for concentrated sample. Vertical lines are as in Fig. 4.

WEINRYB

AND

FIG. G. Circular dichroism spectra (225290 mF) of concentrated (---) and about ten-fold dilute (-----) solutions of Z-Phe-Phe in 71y0 EG. Spectra normalized to equivalent total sample absorbances. Atss.5 rnp = 0.910/mm for concentrated sample. Vertical lines as in Fig. 4.

experimental uncertainty, there appears to be no effect of concentration upon the spectra for the concentration range examined. Profound changes in the CD spectrum result when the phenyl chromophore is incorporated into a dipeptide, as in Phe-Phe (Fig. 5). Negative bands appear and the correspondence of dichroic to absorption bands becomes less clear. Although the spectra of concentrated and dilute dipeptide solutions are similar at higher wavelengths, a significant difference in amplitudes at wavelengths below about 250 rnp is present, and may indicate a concentration-dependent effect on the immediate environment of the phenyl chromophore, although light scattering by the concentrated solution might also partially explain this discrepancy. Experiments with Z-Phe-Phe solutions reveal another qualitatively different type of CD spectrum (Fig. 6). These spectra are characterized by a relatively large negative

STEIKER

band between 230 and 240 rnp lvhich (by comparison with the spectra for Phe-Phe) probably arises from the benzyloxycarbonyl group. The spectra at wavelengths greater than 250 rnp now appear to be wholly negative. Although the wavelength shifts at these higher wavelengths for concentrated vs. dilute Z-Phe-Phe solutions are large enough to be possibly significant in their own right, the most striking concentration-dependent differences are seen below 240 rnp. There the concentrated solution shows a trough at 23s rnp, a 4 .rnF “red-shift” compared to that of the dilute solution. In addition, the negative amplitude of this trough is decreased to some 40% of its dilute solution counterpart; this or may, in fact, reflect the intensification wavelength shift of a positive band for the concentrated solution at still lower \vavelengths. The results of I:ig. 6 suggest that the optical activity of the benzyloxycarbonyl group is particularly sensitive to environmental effects resulting from concentration changes. Examination of the CD spectrum of Z-Phe-Phe in pure EG (Fig. 7) reveals that the negative trough attributed to the

FIG. 7. Circular dichroism spectra (235290 rnp) of concentrated solutions of Z-Phe-Phe in 100~~~ EG. Asss.s rnp = 0.425/mm. Vertical lines as in Fig. 4.

henzyloxycarbonyl group is further redshifted t,o 243-44 rnb and its amplitude further reduced relative to bhe longer wavelength spectral features. This is taken as an indication that the optical activity changes observed reflect changes in the local effective dielectric constant of the environment of the benzylox\-cnrbonyl group. %u.winescence. Significant differences were observed in the phosphorescence lifetimes at 01°K of Phe, Phe-Phe and Z-Phe-Phe (0.7 “C in 71% KG). The values \vere 5.8,4.S, and 3.5 set, respectively. It is uncertain \vhct,her these differences can he attributed in part to varying extents of intermolecular intcracbion. In the case of Z-Phe-l’hc, the contribution of the benzyloxycarbongl group may be a factor in the different lifetimes obtained . The work of Lasha and collaborators (12) indicates, from considerations of the molecula,r exciton model, that aggregation of a syst,em may change its luminescence char,, acteristics. The concentration dependence of quantum yield at 2.5” in 71% EG was examined for the t)hrcc compounds, but the uncertainties arising from the large “inner filter” effect (13, 14) were great enough to mask any significant differences. The fluorescence polarization of Z-I’hePhc in SO5 ethylene gl\-co1 at 25” was also examined. lq‘or excitation at 240 rnM and observation at 300 rnp, the polarization n-as equal to zero, within experimental uncertainty, if gelation ~-as absent. X significant polnrizat8ion (1’ = 0.099) was observed for :L 1.3’; gel in 71’; EC. If t,he gel \\-:w compressed by centrifugation for 2 hr at 30,000 rpm, the supernate shelved zero polarizat,ion. It is thus unlikely that large aggregates (molcculnr weight > 10”) exist’ in equilibrium wit,h the gel (1.5). The preceding sections have presented considerable evidence for a conccntrationdependent self-interaction of phcnylalunyl dipeptides in mixed ethylene glyco-aqueous phosphate buffer solvents. Of particular interest is the striking observation of gel formation for the Z-l’hc-l’he and Alet-l’he dipeptides. Ilinear aggregation of the dipcptidc suburlit)sis a plausible, although conjectural,

explanation of the formation of a gel in these cases. Theoretical considerations suggest that the same hydrophobic and hydrogen bonding interactions which stabilize the helical forms of hiopolymcrs may also permit the formation from their monomer and oligomer units of linear aggregates, which have an ordered structure (16, 17). Several examples of such processeshave been observed corresponding to polynucleotides (18, 19) and polysaccharides (17). In addition, the gelntion of diherlzo~l-I-cystine in nlcohol-water mixtures has been reported (22). It is not yet possibleto propose an explicit model for t,his process. Inspection of spacefilling molecular models appears to allow the possibility that aggregation of Z-Phe-Phc occurs as the result of hydrophobic intcractions of phenyl rings. It is not clear whether the prcferrcd structural lmit of the gel would be a single-chain linear aggregate or a multichain structure stabilized by lateral interactions of the aromat’ic ring systems. Thcsc stem to be among the most plausible possibilities. Examination of molecular models indicates, however, t,hat mutual intercalation of the phenyl rings of tn-o linear aggregates may not be sterically feasible. The role of the solvent system in the format’ion of Z-l’hc-l’hc gels is a closely related problem. The mixed solvent, may partition locally bet\\-ecn the h>-drophohic aromat’ic rings and the ionic carboxJ-1 groups of the peptide ~xd~ho~~c or, altcrnativcly, it may exert its effect on the intcractiou processby virtue of its effcctivc bulk dielectric constant. The ohservat,ion that I’he-l’he does not gel under comp:trable conditions suggests that the ~)etlz~loxyc:lrhon~l group of Z-PhrI’hc stabilizes the aggrcgatcd sq’stcm either by abolishing the positive charge of thcl LYamino group or through the additional hydrophobic intcract~ions of the third phenyl ring. Howwer, the observed gelation of Met1%: indicates that aggregation tloes riot L‘(‘quirc :L multi-ring system. The concentn-ltion-differcllce spectra obtained with a number of phcrlylnlanyl pcptides indicate that self-intrraction is not COIIfined to the systems exhibiting gelation. While a complete study \\-a$ not made, all of the significant difference spectra which \vcre obserwtl occurred in mixed gl)-col-water sol-

270

WEINRYB

AND

vents. The qualitative variations in the difference spectra for different phenylalanyl peptides indicate that the environmental changes which affect the chromophore are dependent upon the chemical nature of the peptide. The possible relevant factors include the mutual interaction of the electron systems of parallel stacked phenyl rings, the influence of a charged site placed in juxtaposition to an aromatic ring (20) and the polarity of the environment (21). From the results of Donovan, Laskowski, and Scheraga (20) it is known that the proximity of a charged carboxylate group produces a red-shift of a phenyl or benzoyl ring spectrum. If the self-interaction of ZPhe-Phe results in the formation of complex species in which a carboxylate group is in juxtaposition to a benzyloxycarbonyl group, then the positive peak at 238 rnp could be explained on this basis. This kind of mechanism is unlikely to be a factor in the difference spectra observed for Leu-Phe and RletPhe, since it is not probable that complex formation would result in a carboxylate site in proximity to the C-terminal phenylalanine, which already bears a charged carboxylate group. For these compounds, a shift of the chromophore to a more non-polar environment, as would accompany the formation of hydrophobic contacts with a leucyl or methionyl group might offer a partial explanation (20). However, since the difference spectra for the above four compounds are wholly positive, they cannot be completely accounted for by a spectral shift but must originate in part from an intensification of the primary absorption band (20). The negative concentration-difference spectra observed for Phe-Leu, I’he-Gly, and Phe-Ala correspond formally to a blue-shift of the primary absorption band, superimposed upon an overall hypochromism. The positions of the negative maxima correspond fairly closely to those wavelengths where the original spectrum has maximum negative slopes (19). The explanations advanced above are clearly not applicable for these compounds. It is possible that the interaction of parallel stacked phenyl rings is important in these cases.

STEINER

The CD spectra of phenylalanine and its dipeptide derivatives are illustrative of the sensitivity of this parameter to changes in the environment of the chromophore. The most prominent feature of the CD spectrum of Z-Phe-Phe, the negative band at 230240 rnp, undoubtedly arises from the benzyloxycarbonyl group. The pronounced concentration dependence of this part of the spectrum suggests that the environment or conformation of this group is modified as a consequence of the self-interaction of Z-PhePhe. The smaller changes observed in the region of the primary absorption band may reflect environmental changes of the phenyl rings. Unfortunately, there is insufficient background information upon the CD spectra of phenylalanine derivatives of known conformation to permit detailed conclusions as to conformation. These data do, however, support the conclusion that selfinteraction of Z-Phe-Phe occurs. REFERENCES 1. NAG-CHAUDHURI, J., AND AUGENSTEIN, L. Biopolymers Symp. 1, 441 (1964). 2. LONGWORTH, J. W. Biopolymers 4, 1131 (1966). 3. BISHAI, F., KUNTZ, E., AND AUGENSTEIN, L. Biochim. Biophys. Acta 140, 381 (1967). 4. STEINER, R. F., AND KOLINSKI, R. Biochemistry 7, 1014 (1968). 5. STEINER, R. F. Biochem. Biophys. Res. Commun. 30, 502 (1968). 6. WEINRYB, I., AND STEINER, R. F. Biochemistry 7, 2488 (1968). 7. HOERMAN, K. C., AND BALEKJIAN, A. Y. Federation Proc. 26, 1016 (1966). 8. FISHER, H. F., AND CROSS, D. G. Arch. Biothem. Biophys. 110, 217, (1965). 9. CROSS, 1). G., AND FISHER, H. F. Arch. Biothem. Biophys. 110, 222 (1965). 10. BEYCHOK, S. Science 164, 1288 (1966). 11. MOSCOWITZ, A., ROGENBERG, A., AND HANSEN, A. E. J. Am. Chem. Sot. 87, 1813 (1965). 12. KASHA, III., RAILS, H. R., AND EL-BAYOUMI, M. A. Pure Appl. Chem. 11.371 (1965). 13. WEILL, G., AND CALVIN, M. Biopolymers 1, 401 (1963). 14. ELLIS, D. W. in “Fluorescence and Phosphorescence Analysis,” (D. M. Hercules, ed.), p. 41. Wiley (Interscience), New York, 1966. 15. WEBER, G. in “Fluorescence and Phosphores-

PROPERTIES

16. 17. 18. 19.

OF

PIIENYLALANYL

cence Aualysis,” (D. ?\I. Hercules, ed.), p. 217. Wiley (Interscience), Sew York, 1966. PETICOLAS, W. L. J. Chem. Phys. 37, 2323 (1962). PETICOLAS, W. L. J. Chew. Phys. 40, 1463 (1964). R.ALPEI, R. K., CONNORS, W. J., AND KHORANA, H. G. J. Am. Chem. Sot. 84,2265 (1962). GELLERT, M., LIPPSET, AI. P;., AND DAVIES,

PEPTIDES

271

D. R. I’TOC. :\‘all. had. Sci. U.S. 48, 2013 (1962). 20. DONOVAN, J. W., IASI;OWSKI, JR., hf., AKD SCHEI~AGA, II. A. J. Am. Chem. Sot. 83, 2686 (1961). 21. YANARI, S., AND IJo~EY, F. J. Biol. Chew 236, 2818 (1960). 22. GORTXER, R. A., ANO IIOFFMAN, W. I?. J. Am. Chem. Sot. 43, 2199 (1921).