The Spectrophotometric Titration of Polyacrylic, Poly-L-aspartic, and

RUTHMCDIARMID AND PAUL DOTY

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The Spectrophotometric Titration of Polyacrylic, Poly-L-aspartic, and Poly-L-glutamic Acids’

by Ruth McDiarmid and Paul Doty

Downloaded by UNIV OF MANITOBA on September 7, 2015 | http://pubs.acs.org Publication Date: August 1, 1966 | doi: 10.1021/j100880a030

Department of Chemistry, Harzard University, Cambridge, Massachusetts 08138

(Received February 11I 1966)

The p H dependence of the far-ultraviolet spectra of polyacrylic, poly-L-aspartic, and polyL-glutamic acid was examined in order to delineate how carboxyl ionization and chain configuration affect the spectra of linked carboxyl and peptide groups. The spectrophotometric titration of polyacrylic acid differed from acetic acid in that the extinction coefficient near 185 mp was less than proportional to the degree of ionization. This effect was not apparent in the polypeptides. Poly-L-aspartic acid displayed no obvious configurational change as a function of ionization, but the results were not inconsistent with the assumption of some short helix formation. Its increase of absorbance with ionization was consistent with that of simple carboxylic acids at 185 and 190 mp but was greater a t higher wavelengths, indicating a charge-induced alteration of the peptide absorption. By contrast, the extinction coefficients of poly-L-glutamic acid increased somewhat more strongly a t low and high degrees of ionization and, more importantly, displayed a further substantial rise at intermediate degrees of ionization corresponding to the helix-coil transition. The rise due to the helix-coil transition was quite dependent on ionic strength, increasing, for example, from 1600 to 2650 at 190 mp as the counterion concentration was reduced from 2.0 to 0.0004. This variability, together with a 4-mp shift of the poly-Laspartic acid spectrum compared to poly-clysine, shows that the peptide bond in the randomly coiled configuration displays a range of characteristic properties, unlike the helical form where an essentially invariant behavior is observed.

This investigation is focused on the effect of chain conformation and carboxyl ionization on the far-ultraviolet spectrum of poly-L-glutamic acid (PGA). The motivation for this work lies in the following circumstances. I n 1957 it was found that PGA in aqueous solution exists in the form of rod-like helices when it is largely untitrated (below pH 4.7 in 0.2 M NaC1) and as a randomly coiled chain when predominantly ionized (above pH 5.5 in 0.2 M NaCI).2 Somewhat later it was shown that the helical form was considerably hypochromic to the coil form in the absorption band near 190 mp3 and that the spectrum of the helical form displayed a characteristic ~ p l i t t i n g . ~I,n~ 1962 the possibility of using this change in extinction as a measure of helix content was considered unlikely6-8 for several reasons: the ionization of the carboxyl groups may perturb the peptide bond adsorption; The Journal of Physical Chemistry

the transition between the two differently shaped adsorption bands may not be linear with helical content owing to the different shapes; and changes in optical rotation found on titrating y-poly-D-glutamate, which cannot take on a helical conformation, may have their (1) This work was supported by a grant (GB-1328) from the National Science Foundation and by fellowships to R. McDiarmid from the National Science Foundation and the National Institutes of Health. (2) P. Doty, A. Wada, J. T. Yang, and E. R. Blout, J . Polymer Sci., 23, 851 (1957). (3) K. Imahori and J. Tanaka, J . Mol. Biol., 1 , 359 (1959). (4) P. Doty, J . Polymer Sci., 49, 129 (1961). (5) K. Rosenheck and P. Doty, Proc. Natl. Acad. S c i . , U . S., 47, 1775 (1961). (6) A. Wada in “Polyamino Acids, Polypeptides and Proteins,” M. A. Stahmann, Ed., University of Wisconsin Press, Madison, Wis., 1962, p 157. (7) J. Applequist, ref 6, p 158. (8) T. Miyazawa, ref 6, p 159.


TITRATION OF POLYACRYLIC, POLY-L-ASPARTIC, AND POLY-L-GLUTAMIC ACIDS

counterpart in spectral change^.^ Moreover, the first attempts to correlate the extinction coefficient near 190 mp and the helix content of proteins was only partially successfu1.j Hence, the need to examine the absorption in the ultraviolet as a function of carbonyl ionization, interactions between neighboring carboxyl and peptide groups, and chain conformation was fairly evident. I n order to differentiate the possible interactions involved it seemed desirable to investigate two other polymers. One was polyacrylic acid, chosen because it contains carboxyl groups as the only functional and chromophoric groups and therefore permits the assessment of any consequences of carboxyl group interaction. The other was poly-L-aspartic acid. This was selected because it provides a chain in which the carboxyl and peptide groups are similarly interacting as in PGA and yet exhibits little secondary structure. Indeed the uncertainty of the extent to which it takes up a helical configuration provided a secondary incentive for including it. The actual investigations of this polymerlOjllhave concluded that in the nonionized form it is about 20% helical, but our initial view was that this could well have been due to perturbation effects and that it may indeed be devoid of helical structure. In any event, it furnishes a substance that has very much less secondary structure than PGA.

Experimental Details Materials. Polyacrylic acid was a gift of the Union Carbide Chemicals Corp. It had a weight-average molecular weight of about 80,000. Poly-L-aspartic acid, mol w t ca. 25,000, was purchased from Pilot Chemicals Go., Waltham, Mass. Poly-L-glutamic acid was made from the benzyl ester obtained from the same source. The PGA had a molecular weight of about 80,000. Neither polyamino acid displayed any adsorption in the 280-mp region. All polymers were dissolved in water, the pH adjusted to 7, dialyzed, and lyophilized. NaC104 and Mg(C104)2,supplied by G. Frederick Smith Chemical Co., were purified by crystallization from water. The perchlorates were used in place of the more usual chlorides because of their transparency below 200 mp. Baker and Adamson reagent grade HC104 was used in the spectral work for the same reason. Concentration Determinations. The concentration of polyacrylic acid was determined by titrating a stock solution of the polymer to the phenolphthalein end point. The concentrations of polyaspartic and polyglutamic acids were determined by micro-Kjeldahl analyses of the stock solutions. I n all cases, the determinations were repeated several times ; the maximum deviation was within 297, of the stated concentra-

2621

tions. All concentrations are expressed in terms of the concentration of carboxyl groups, in moles per liter. Potentiometric Titrations. Polymer solutions containing approximately 0.003 mole of carboxyl groups/ 1. were titrated a t 25" under nitrogen. The titration was conducted either automatically, with a Radiometer T T T l a titrator equipped with a G202B (high pH) glass electrode and a K4312 reference electrode in conjunction with a SBUl syringe buret and a SBR2a recorder, or manually using the same pH meter and electrodes. In either case, previously calibrated 0.2ml syringe burets were used. Only a base titration was performed on polyacrylic acid; only acid titrations were performed on polyaspartic and polyglutamic acids. Spectrophotometry. Spectra were measured in a Beckman DK-2A ratio recording spectrophotometer that was rapidly flushed with nitrogen when used below 200 mp. By the use of 2-mm cells and the nonabsorbing salts and acid of clod-, the optical density of the reference cell was kept to less than 0.2 through this investigation. In the spectral work, the residue concentrations were approximately 3 X M , polyM , polyaspartic acid; and 3 X acrylic acid; M , polyglutamic acid. I n the spectrophotometric titrations, the pH of the sample could not be measured before its spectrum was recorded because C1- leaking from the saturated calomel electrode altered the absorption below 200 mp. Instead, for polyacrylic and polyaspartic acids, aliquots of a solution previously brought to the desired concentration and maximum or minimum pH were placed in a series of stoppered test tubes, a small quantity of HC1O4 or NaOH was added to one test tube, the spectrum of its contents was measured, and its pH was determined. This was repeated until the entire titration curve had been investigated. A parallel titration was conducted on the solvent, which was used as the reference solution for the spectral measurements. Although, by this procedure, the reference solution contained the same perchlorate concentration as the sample, the two were generally a t different pH values. I n the basic region where, because of hydroxyl absorption, two solutions a t different pH values absorb differently, this spectral difference was corrected by means of a previously determined graph of the absorption of OH- as a function of concentration, in the same cells. (9) H. Edelhoch and R. E. Lippoldt, Biochim. Biophys. Acta, 45, 205 (1965). (10) G . Spach and J. Brahms, Nature, 200, 72 (1963). (11) A. L. Jacobson, Biopolymers, 3, 249 (1965).

Volume 70, Number 8 August 1966


2622

Table I : Molar Extinction Coefficient Data for Various Acids 185 Acid

Acetic Polyacrylic Polyaspartic (0.001 M ) Polyglutamic (0.0004 'Vf)

-

X, m p

c

ea-0

de/da

75 1i o 6130

2670 1800" 3140 2420

3800

3400*

200

195

190 dc/da

ea- 0

fa-0

ddda

(a-0

de/do

40 75

345 430

...

...

...

100

6520

940 900" 1100 1100

5640

965

3980

980

4400

1400*

3650

14OOb

2575

1400*

40 80


These are the initial and terminal slopes, Le., a t a = 0 and CY = 1. * These are average values for the initial and terminal slopes and do not take into account the additional increase that occurs in the central part of the titration due to the helix-coil transition.

For polyglutamic acid, the above method could not be used because small amounts of the polymer precipitate a t the site of acid injection. Instead, the entire sample was put in a stirred vessel and a small quantity of HC104 was added; an aliquot was removed, its spectrum was measured, and its pH was determined. This was repeated until the entire titration curve of polyglutamic acid had been investigated. As before, a parallel titration was conducted on the solvent; compensation was made for spectral differences arising from differences in pH.

Results The titration curve of acetic acid was measured potentiometrically and followed spectrophotometrically to determine whether the spectral manifestations of the carboxyl-carboxylate conversion in a noninteracting system are, in fact, linear in our representation. At 185, 190, and 195 mp, the absorption was indeed found to depend linearly on the degree of ionization, a, of the acetic acid solution. The slope of e vs. a for each wavelength are presented in the first row of Table I. Polyacrylic Acid. The potentiometric titration curves of polyacrylic acid displayed the characteristic features noted before.I2 The first ionizations occur a t a pH predicted by the intrinsic pK of the ionizing group. The apparent pK increases as the polymer is further ionized because a greater amount of electrostatic work is needed to remove a proton from the increasingly net negative field produced by the ionized carboxyl groups. An increase in the ionic strength reduces the range of pH over which the titration occurs. I n the modified potentiometric titration curves, in which pH-log [a/(l - a)] is plotted against a, these features take the form of gradually decreasing slopes and downward displacement with increasing ionic strength. The dependence of polyacrylic acid absorption a t The Journal of Physical Chemistry

a Figure 1. Molar extinction coefficients of polyacrylic acid as a function of degree of ionization a t three wavelengths: 0, 0.003 M residues unbuffered; 0, in 0.01 M NaClOa; 8 , in 0.2 M NaC104.

185, 190, and 195 mp on CY in solutions ranging from no added salt to 0.2 M is displayed in Figure 1. We note, first, that the ionic strength of the medium has no influence on E a t a given a. Next we note that, unlike the case of acetic acid, there is a marked curvature for e 185 vs. a ; it is less a t 190 and is absent a t 195 mp. Poly-L-aspartic Acid. The potentiometric titration ~

~~

(12) See, e.g., A. Katchalsky, J . Polymer Sci., 7 , 393 (1951).

2623


I

-I 8000

7000

6000

E


mnn

L

-I

F

3000 0

I

.2

.4

.6

.8

1

a Figure 3. Molar extinction coefficient of poly-baspartic acid as a function of degree of ionization, in 0.2 M NaC104. Figure 2. Modified potentiometric titration curves of poly-a-baspartic acid: 0, 0.001 M residues, unbuffered; 0, in 0.01 M NaClO4; 8, in 0.2 M NaC1O4; (3, in 2.0 M NaC104.

curves of polyaspartic acid are presented in the modified form in Figure 2. These are roughly similar to those of polyacrylic acid in that the slopes diminish and the curves are displaced downward with increasing ionic strength. The curvature, particularly at 0.01 M , is, however, more pronounced in the case of poly-^aspartic acid. The behavior in water is almost identical with polyacrylic acid except that the latter is displaced upward by 0.8 pH unit. Except for the curvature a t 0.01 M there is no evidence of a conformation transition here, and the 0.01 M result is very likely a consequence of the nature of the polyion expansion with charging. The dependence of polyaspartic acid absorption a t four wavelengths on a is displayed in Figure 3 for the case of 0.2 iM NaC104. The results obtained in no added salt and 0.01 M are essentially the same. We see that the absorption increases linearly with increasing degree of ionization of the polymer; there is no curvature and there are no discontinuous breaks in the plots. This linearity clearly indicates an absence of a conformational transition. Examination of the averaged values of the slopes and intercepts listed in Table I show normal values for the slope a t 185 and 190 mp. At 195 and 200 mp, however, the value of the slope remains a t about 1000, rather than falling as it did for acetic and polyacrylic acid. This difference

occurs in PGA as well. It evidently represents a carboxyl-peptide interaction that must be taken into account. The very much higher values of the intercepts are, of course, due to peptide bond absorption. A further point becomes evident upon looking a t the individual values for the slopes and intercepts of e vs. a. These are assembled in Table 11. It is seen that the slopes are essentially independent of ionic strength but that there is a systematic increase in the intercept with decreasing counterion concentration. This suggests that the extinction in the peptide band is substantially more a t lower ionic strengths. This is unlike the behavior of polyacrylic acid. We will return to this point after examining the situation in PGA. Poly-L-glutamic Acid. The modified titration curves a t two concentrations in water and three salt concentrations are shown in Figure 4. Since a helix-coil transition occurs in each of these cases, it is evident that it is more clearly expressed the lower the ionic strength. This is particularly true in the aqueous titrations despite the variable counterion concentration. At 2.0 M there is almost no effect of the transition left in the modified titration curve. The results at three salt concentrations are similar to those of Nagasawa and H01tzer'~but differ from the latter in their apparent dependence of pKo on ionic strength. This is in contrast to other comparable results14which display a somewhat different shape. (13) M. Nagasawa and A. Holtsar, J. Am. Chem. Soc., 86, 538 (1964).

Volume 70,Number 8 August 1966


2624

Table I1 : Spectrophotometric Titration of Poly-baspart'ic Acid NaC104


A,

concn,

mfi

M

ea-0

dr/da

185

0.001 0.01 0.2

6327 6037 6020

2038 2855 2358

190

0 001 0.01 0.2

6687 6517 6354

1010 1366 1070

195

0.001 0.01 0.2

5802 5652 5448

957 1241 920

200

0 IO01 0.01 0.2

4111 3981 3794

1004 1255 904

t

A titration in the presence of 0.01 M Mg(C104)Z yielded a nearly flat modified titration curve having values lying between 4.9 and 5.1. Thus the divalent ion is about two orders of magnitude more effective in suppressing the change of electrical free energy with charge as well as the effect which the helix-coil transition has on this quantity. This is consistent with the findings of Jacobson." The spectrophotometric titrations are shown in Figure 5 for four salt concentrations and a t three wavelengths. I n contrast to poly-L-aspartic acid and polyacrylic acid, the variation of E with CY is not linear or nearly so; instead the curves begin and end with linear regions of positive slope that are joined by a steeper region. Further inspection shows that the two linear regions have essentially the same slope (1300 to 1500) except for the case of 185 mp where the data are quite limited. The spectrophotometric titration curves, while more complex than for the other two polyacids, have a simple interpretation. The initial rise in e with CY is similar to that for poly-L-aspartic acid; then the helix-coil transition causes a more rapid rise, the center of which moves from CY = 0.4 to 0.55 with increasing ionic strength; and finally there is a return to the initial slope once the helix-coil transition is complete. I n order to examine the data in Figure 5 in more detail, it is useful to impose upon the data the assumption that the slopes of the initial and final linear regions are the same for all three wavelengths: 190, 195, and 200 mp. Taking the average value of 1400 for the slope, this cont,ribution has been subtracted from the E value of Figure 5, and the results plotted in a difThe Journal of Physical Chemistry

Figure 4. Modified potentiometric titration curves of poly-a-bglutamic acid: and A, 0.0004 M residues, unbuffered; o----O,003 M residues, unbuffered; 0, in 0.01 M NaC104; 8 , in 0.2 M NaCIO,; (3, in 2.0 M NaClO,.

ferent grouping in Figure 6. Here the results for each wavelength are plotted on common axes. As a result we see more clearly that the transition moves to higher CY values and broadens with increasing ionic strength, but the more novel conclusion is that the change in extinction coefficient for the helix-coil transition increases by more than 60% with decreasing ionic strength. This variation of Ae for the helix-coil transition is seen more clearly in Table I11 where, for each of the three wavelengths, values of e for CY = 0 and values of E extrapolated back to CY = 0 from the high CY region, using the slope value of 1400, are presented. Thus the difference of these given in the fourth column is the estimated change, AE, for the helix-coil transition since the effect of carboxyl ionization has been approximately removed. It is seen that the values rise from 65 to 100% as the ionic strength decreases from 2.0 to approximately 0.0004 M. At all ionic strengths the maximum change occurs a t 195 mp. We note further that a t each wavelength the e for the helical form is approximately independent of ionic strength; the variations arise predominantly in the randomly coiled form. Put another way, 8196 increases above a value of 3650 for the helix by an amount that varies from 45% at 2.0 M to 75% a t about O.OOO4 M . ~

(14) A. Wada, Mol. Phys., 3 , 409 (1960).


2625


Table I11 : Spectrophotometric Titration of Poly-L-glutamic Acid

Counterion concn, M

a = O

Extrapolated from high atoa = 0 (assumed slope)

0.0004 0.003 0.01 0.2 2.0

4700 4600 4330 4380 4420

0.0004 0.003 0.01 0.2 2.0 0.0004 0.003 0.01 0.2 2.0

A --

Coilhelix

From measured slopes

190 m p 7190 7260 6400 6360 6050

2490 2660 2080 1980 1630

2120 2110 1970 1790 1570

Figure 6. Peptide bond absorption of poly-a-bglutamic acid: (1) in 2.0 M NaC104; (2) in 0.2 M NaC104; (3) in 0.01 M NaC104; (4) 0.003 M residues, unbuffered; (5) 0.0004 M residues, unbuffered.

2840 3750 3500 3670 3660

195 mp 6600 6620 5850 5740 5350

2750 2970 2350 2070 1690

2680 2640 2170 2000 1450

2760 2650 2500 2550 2550

200 mp 4840 4770 4210 4050 3700

2080 2120 1710 1500 1150

2230 2160 1700 1390 1010

The best estimate of the individual initial and terminal slopes have also been compiled, and the values of Ae for the helix-coil transition are given in the last column of Table 111. The same effect with ionic strength is seen, and hence the foregoing conclusions apply, independent of the assumption that de/da: = 1400. Spectrophotometric titrations carried out in 0.01 M hIg(C104)2 were similar to the results in Figure 5 for 2.0 M Naf. Thus they showed the suppression of the change in extinction coefficient with the helix-coil transition that was expected in view of the damping out of the effect in the modified titration curve. Xumerically the change in E for the transition was about 1000, substantially less than that found in 2.0 M IfaClOI (except at 200 mp).

Discussion

a

Figure 5. Molar extinction coefficient of poly-a->glutamic acid as a function of degree of ionization a t several wavelengths: 0, 0.003 M residues, unbuffered; 0, in 0.01 M NaC104; 8, in 0.2 M NaClOa; @, in 2.0 M NaC104.

We are now in a position to review and compare the contributions which each of the polymers studied has made to our main concern of how the ultraviolet absorption of the polypeptide chain is affected by carboxyl ionization itself and the possible charge-induced conformational changes that this may bring about. Carboxyl-Carboxylate Ion Absorption. The absorption due to the uncharged carboxyl group is almost negligible; its molar extinction coefficient rises from essentially 0 at 200 mp to about 100 at 185 mp. Upon conversion to the carboxylate ion, its extinction coefficient rises to 500 at 195 mp, 1050 a t 190 mp, and 2400 a t 185 mp. These values cannot be determined directly in the acidic polypeptides examined here because of the enormous absorption of the peptide bond in this region. However, the change in extinction coefficient with degree of ionization (the slope, dO/da:) is consistent with these values a t 185 and 190 mp. On the other hand, at 195 and 200 mp the slope remains a t about 1000 instead of decreasing sharply as it does Volume 70, A'umber 8

August 1966

RUTHMCDIARMIDAND PAULDOTY


2626

in polyacrylic and acetic acids. Whether the absorption a t these higher wavelengths is due to a broadening of the carboxylate bands or a charge-induced effect in the peptide group cannot be unambiguously stated although the latter is more likely. An anomaly was found in polyacrylic acid in that the initial increase of E with a diminished with titration. This curvature in the e vs. (Y plot, evident at 185 and 190 mp, was not apparent in the polypeptide titrations; it was judged to be due to inductive effects between closely connected carboxylate groups inasmuch as it was not ionic strength dependent. Hence it was of no further concern. Poly-L-aspartic Acid. The interpretation of the titration data on poly-L-aspartic acid requires a decision on whether or not detectable amounts of the helical conformation form a t low pH. There are three strong indications in the work presented here that polyL-aspartic acid remains in the randomly coiled form a t all degrees of ionization. The first is the absence of any pronounced maximum and minimum in the modified potentiometric plots (Figure 2). The curvature found here is only slightly more than that found for polyacrylic acid. The second indication is the linearity displayed by the spectrophotometric titration plots (Figure 3). Assuming that the hypochromic effect is well established, and the observations on poly-Lglutamic in this work support this, even a few per cent helix formation would have been detectable in deviations from linearity in Figure 5 and similar plots obtained a t the other ionic strengths.15 Finally, there is the direct observation of the extinction coefficient a t 190 mp, probably the most reliable wavelength for this purpose. The average value found here for poly-Laspartic acid, 6520, is close to that for poly-L-glutamic acid in the coiled form, 6650 by this study, and to that of poly-L-lysine in the coiled form, 6900.5 Thus, this evidence would suggest that poly-L-aspartic acid is in the randomly coiled form when nonionized, as well as throughout its titration range. However, this conclusion is in some conflict with the rather meager optical studies on this polymerlO!ll which suggest that it is about 25% helical in acid solution. The only obvious way in which both this and the above conclusions could be consistent is if the helical regions were short and self-limiting, that is, unable to develop long-range structure. If this were the case, they would be gradually eliminated upon titration without displaying a helix-coil transition. Moreover, the apparent agreement of the extinction coefficients could possibly be misleading because of uncertainties in the concentration of counterions.lB On the other hand, the optical studies are far from comThe Journal of Physical Chemistry

1 1000

-

04-

I80

I

I99

210

200

'220

230

X(rnp)

Figure 7. Random-coil peptide bond absorption spectra: -, poly-baspartic acid, p H 3.05; 0, poly-baspartic acid, a = 0 (Table 11); 0 and 0, poly-clysine (from Rosenheck and Dot,y6).

plete. Hence, at present one can only conclude that there is no long-range helical structure in poly-Laspartic acid, but perhaps as much as 25% of this form may be present in short helical regions which do not display a cooperative helix-coil transition. Proceeding on the basis that there are no extensive or sharp conformational changes occurring within the titration range of poly-L-aspartic acid, our conclusions on its spectrophotometric behavior are quite simple. The ultraviolet absorption band for the acid form is found to decrease somewhat with ionic strength, an observation we will return to below. Upon titration the absorption rises, while a t 185 and 190 mp the rise a t moderate ionic strength is that predictable from the change in extinction coefficient upon ionization of the carboxyl groups in acetic or polyacrylic acid. However, at 195 and 200 mp, the extinction coefficient rises considerably more with ionization than is found for carboxylic acids. This suggests that the peptide absorption is enhanced on the high-wavelength side by neighboring carboxylate ions. A remaining question is whether or not the ultraviolet absorption spectra for poly-L-aspartic acid is the same as that for the other available spectrum of a randomly coiled polypeptide chain, poly-L-lysine in neutral s ~ l u t i o n . ~The comparison is made in Figure 7 where the spectrum of poly-L-aspartic acid a t pH 3.05 is presented together with representative spectra of (15) R. S. McDiarmid, Ph.D. Thesis, Harvard University, Cambridge, Mass., 1965. (16) .Current work (Sarkar and Doty) shows the 6 for poly>lysine in the coil form to be very dependent on counterion and indicates that the published values6 may be too low.



poly-L-lysine from earlier The values of e at a = 0 for the wavelengths studied in this investigation are included, and they are seen to fit on the spectrum. It is evident that there is a blue shift of about 4 mp and a broadening of the poly-L-aspartic acid spectrum relative to the poly-L-lysine. As this effect appears representative of the various amino acids in peptide form,” it makes a strictly quantitative interpretation of this band for proteins rather difficult. I n this case, the origin of the shift could be either a perturbation of the peptide transition by the charged lysine groups or inductive effects arising from substitution of the p carbon in the poly-L-aspartic acid. I n any event the spectrum of the peptide group is seen to be significantly dependent on the amino acid residue to which it is attached, and this will have to be taken into account in any quantitative applications. Poly-L-glutamic A c i d . The spectrophotometric titrations of PG14 display the striking rise accompanying the helix-coil conversion. Otherwise they show essentially the same features noted in poly-L-aspartic acid. Outside the transition range, the slopes of E us. a are about the same. Their magnitude is, however, somewhat larger than poly-L-aspartic acid. In analyzing the situation according to Table I11 it was seen that a consistent picture results from taking 1400 as the slope of E us. a for all the initial and terminal regions of the plots in Figure 5. This value is seen to be somewhat higher than the corresponding values for poly-L-aspartic acid in Table I1 (900 to 1370). While the analysis in Table I11 could have been carried through nearly as well with a constant value of 1300, the use of the range of values found for poly-L-aspartic acid would have meant misfitting a considerable part of the data. Thus this represents a small but real difference in the behavior of the two polypeptides. Kevertheless, polypeptides demonstrate what must be accepted as a firm conclusion: that the peptide absorption band for the randomly coiled form of polypeptides

2627

displays a hyperchromic shift with the reduction of count erions. This hyperchromic shift as seen in Table I11 is substantial (65 to 100%) compared to the change in E due to the helix-coil transition. It is probably due to a charge-induced perturbation that becomes more intense as the counterion shielding is reduced. The result is that the randomly coiled form must be considered to have a variable extinction in the polypeptide band, although the variation observed here is probably rather extreme compared to differences in environments that might occur in protein molecules. Kevertheless, this effect, taken together with the wavelength shift discussed in the preceding section, defines a range of variation in the absorption spectrum of polypeptide chains that is substantial. As a result, quantitative use of this band in analyzing proteins for conformations is not possible, although useful semiquantitative information may be expected. More generally it appears that the somewhat variable spectroscopic properties of the peptide bond in the form of randomly coiled or disordered polypeptide chains must be taken as a characteristic of this conformational state. That is, all the optical properties associated with the peptide bond must be considered somewhat variable in the absence of secondary structures such as the a helix which imposes a more constant and rigid environment on the peptide bond. I n the future it will be useful to delimit the range of these properties which the peptide bond in this form can display to those likely to occur in protein structures. Such a narrowly defined “standard state” would make more precise and reliable the information which optical methods can provide on protein conformations. Acknowledgment. We are particularly indebted to Drs. Walter Gratzer and George Holzwarth for discussions and advice during the course of this investigation. (17) A. Schmitt and G. Siebert, Z . Phgsiol. Chem., 335, 187 (1964).

Volume 70,)\-umber 8

August 1966

The Spectrophotometric Titration of Polyacrylic, Poly-L-aspartic, and

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