interpretation of thermal perturbation spectra of proteins

Int. J. Peptide Protein Res. 10, 1977, 71-79 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

INTERPRETATION OF THERMAL PERTURBATION SPECTRA O F PROTEINS

J A K E BELL0

Department o f Biophysics, Roswell Park Memorial Institute, Buffalo, New York, U.S.A.

Received 21 December 1976, accepted for publication 21 January 1977

The thermal perturbation difference spectrum of reduced lysozyme has a long wave length extremum at 304 nm a t pH 6.1 S and a very small extremum at 306 nm at pH1.S. These results differ from those o f Leach & Smith (1972), which showed an extremum at 293 nm. the same as for model tryptophyl compounds. Our result may arise from a conformational difference between the two sample temperatures. The interpretation o f thermal perturbation spectra o f proteins is discussed. Contributions from thermally induced concentration differences, buried chromophores, and chromophores in crevices are considered in the interpretation of the thermal perturbation spectrum o f bovine serum albumin.

It is suggested that chromophores in pauciaqueous crevices may appear buried toward thermal perturbation spectroscopy but accessible toward solvent perturbation and chemical reagents.

Leach & Smith (1972) and Nicola & Leach (1976) have published thermal perturbation (TP) spectra of several proteins, among them lyoszyme, reduced lysozyme and bovine serum albumin (BSA). This communication deals with differences in the TP spectra of reduced lysozyme as reported by these authors and by us (Bello, 1970) and with the interpretation of TP spectral data, in particular as related to bovine serum albumin (BSA).

RESULTS AND DISCUSSION

Reduced lysozy me We have published thermal perturbation difference spectra of lysozyme and reducedcarbox- amidomethyllysozyme ( RCAM - lysozyme ) (Bello, 1970). Leach & Smith (1972) published similar experiments for lysozyme and reduced- carboxymethyllysozyme (RCM -1ysozyme). (Note the difference in the sulfhydryl blocking group, carboxamidomethyl and carboxymethyl.)

For native lysozyme the results of both labora- tories are substantially alike. But for reduced lysozyme they are quite different. We reported that RCAM-lysozyme showed a negative extremum at 303nm instead of the 293nm extremum shown by N-AcTrp-NH2 and by RCAMchymotrypsinogen. Leach and Smith reported that RCM-lysozyme has a negative extremum at 293nm. (Leach & Smith (1972) showed a thermal perturbation spectrum of L- tryptophan with the major negative extremum at 283 nm. Correspondence with Dr. Leach has indicated that the wave length scale of their Fig.4b should be shifted lOnm to the left. That is, the extremum for tryptophan is at 293 nm, as it is for N-AcTrp-NH2 (Bello, 1970). Dr. Leach states that the wave lengths mentioned in the fext of h a c h & Smith (1972) are correct; they refer to the spectrum on the correct wave length axis.) We dissolved our RCAM-lysozyme in 0.025 M ammonium acetate at pH6, while

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1 1 I I

260 280 300 320 I I

WAVELENGTH, nm FIGURE 1 Thermal perturbation spectra of lysozyme and RCAM- lysozyme. 1) RCAM-lysozyme, 1 mg/ml, pH 6.12,26" vs. 4"; 2) RCAM-lysozyme, 0 5 mg/ml, pH 1 .5,26" vs. 4"; 3) RCAM-lysozyme, 05 mg/ml, pH15, 27" vs. 17"; 4) RCAM-lysozyme, 0.33 mg/ml, pH6.15, 26" vs. 4" ; 5) native lysozyme, 05 mg/ml, pH 1 5 . Solutions at pH 15 were adjusted with HCI and contain 0.15 M KC1; solutions at pH6.15 contain 0.025 M ammonium acetate. The magnitudes of the extrema of curves 1 and 4 are not proportional to concentration.

the solvent used by Leach and Smith was 0.15 MKCl at pH 153.

We have repeated our experiments with one of the earlier batches and with a new batch of RCAM-lysozyme, and obtained substantially the same result as before. In Fig. 1 we see that RCAM-lysozyme at pH 6.1 has a negative extremum at 304 nm, at concentrations of 1 mg/ ml and 033mg/ml. At pH1.5 in O.1SMKCl our RCAM-lysozyme shows a very slight negative extremum at 306nm; this occurs when the temperatures are 27" vs. 4" and 27" vs. 17". We see no negative extremum at about 293 nm for RCAM-lysozyme at pH 1.5. We also prepared RCM-lysozyme (sulfhydryl groups alkylated with bromoacetic acid). At pH1.5 this gave a spectrum hardly distinguishable from that of RCAM-lysozyme , except that the negative extremum was at 305 instead of 306 nm.

At pH 1.5, native lysozyme has a negative extremum at 294nm, with a shoulder at about 300nm. This spectrum is rather similar to that shown earlier (Bello, 1970) for native lysozyme

BELL0

at pH4.1. Aezw corresponds to 2.1 exposed tryptophyl side chains, in substantial agreement with the result of Leach and Smith. This result must be considered with caution because the model compound, N-AcTrp-NH2, does not show a shoulder at 300 nm. The 300 nm shoulder for native lysozyme might arise from tryptophyl in a special environment. We found no difference in results with lysozyme supplied by Calbiochem or by Worthington. The spectrum of N-AcTrp- NH2 at pH 1.5 is almost indistinguishable from that at pH 6.1 .

The 303nm extremum for RCAM-lysozyme at pH 6 raises the possibility that one (or more) tryptophyl residue is in a special environment. The absence of a negative extremum at 293 nm and the presence of a large positive extremum at 293 nm for RCAM-lysozyme at pH 6 suggests a conformational change on cooling to 4", with burial of tryptophyl residues. An even larger conformational effect may occur at pH 1.5. (Note that spectrum 2 is at one-half the concentration of spectrum 1 .) The resulting red-shift would cancel much of the negative extremum leaving only the remnant observed at 303-306 nm. There need not be any tryptophyl residues in special environments. Native plactoglobulin and a-chymotrypsinogen show negative extrema at 303nm in addition to extrema at 293nm (Bello, 1970). The 303nm extrema of these proteins appear to be of a different character from that of RCAM-lysozyme, and may arise from special tryptophyl residues. Also, RCAM- a-chymotrypsinogen has a negative extremum only at 294nm, with an integrated intensity about equal to the sum of the two extrema of the native protein. Reduced P-lactoglobulin was not studied.

Our earlier report (Bello, 1970) had shown that RCAM-lysozyme in 6 M Gdn * HCl (guanid- inium chloride) gives the thermal perturbation spectrum expected for an unfolded protein with nearly six exposed tryptophyl residues. Our result for unreduced lysozyme (Bello, 1970) in 6 M Gdn- HCI indicated nearly complete exposure of six tryptophyl residues, while Leach and Smith found only 3.6 for lysozyme in 8MGdn.HCI. Leach and Smith suggested that the discrepancies arise from "processing of the data", or in plainer words, that we made mistakes in calculation. Reexamination of our

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data shows this to be partly the case. In our Table 1 (Bello, 1970), is shown exposure of tryptophyl in 6MGdn-HCI calculated at 293 nm and at 300 nm (the latter in order to reduce interference from tyrosyl). At 300 nm our calculations are correct, giving 5.6 and 5.9 exposed tryptophyl residues for lysozyme and RCAM-lysozyme respectively. But at 293 nm our calculations are wrong. The correct values should be 4.5 for lysozyme and 5.1 for RCAM- lysozyme, instead of the earlier 5.4 and 5.6. In our earlier work (Bello, 1970) we had given reasons for considering our lysozyme (but not RCAM-lysozyme) to contain about 10% of impurity not contributing to the spectrum. This correction would raise the exposed tryptophyl estimate to 5.0 for lysozyme in 6MGdn-HCl The values (for 27” us. 3”) were given as 492 for lysozyme and 5 10 for RCAM-lysozyme. The correct values are 413 (1721deg) for lysozyme and 471 (19.6/deg) for RCAM-lysozyme.

We have now estimated the exposed tryptophyls from the integrated intensity of the 293 nm lobe. For native lysozyme at pH6, the number of exposed tryptophyl residues is 2.8 (3.1 with a 10% adjustment). For Gdn-HCI solutions the number of exposed tryptophyls is 4.9 for lysozyme (5.3 with a 10% adjustment) and 5.2 for RCAM-lysozyme. Our value for lysozyme is considerably larger than the 3.6 found by Leach and Smith for 8 M Gdn. HCI.

The substantial difference between the calculated exposures at 300 and 293nm for lysozyme and RCAM-lysozyme is puzzling because the contribution from tyrosyl in 6 M Gdn. HC1 is much smaller at 300 nm than at 293 nm (Bello, 1969). The tyrosine contribution to the exposure calculated from the integrated intensity is small, since lysozyme has only one- half as many tyrosyl as tryptophyl residues (Canfield, 1963), and the thermal perturbation spectrum of tyrosyl in 6MGdn.HC1 has an integrated intensity only one-tenth (Bello, 1969; 1970) as large as that of tryptophyl over the wave length range of the negative lobe of the tryptophyl difference spectrum (with extremum at 293-294 nm).

Thermally induced comefitration differences Since a TP difference spectrum uses two temperatures, there is induced a density difference

and, therefore, a chromophore concentration difference. Since the solution in the “sample” beam is normally colder than that in the ‘Lreference” beam, the former normally has the higher concentration. This results in the super- position of a fractional amount of the direct spectrum on the TP spectrum, resulting in too high a A€ in the region of positive A€ and too small IAEI in the region of negative Ae. For water, the concentration difference is about 0.27% for the 15” temperature difference used by Nicola and Leach. For organic solvents the effect is greater. When all of the chromophores of a protein are well exposed, the concentration effect introduces no error in the estimation of exposed groups, since the chromophores of both the protein and model are equally affected. But when some of the protein chromophores are buried, the concentration effect becomes significant. The error introduced can be eliminated by a 4cell method in which the warm compartment contains an additional cell with a solute concentration equivalent to the excess concentration of the cold cell. The cold compartment contains an extra solvent cell. An example of a 4cell procedure for phenylalanine has been published (Bello & Bello, 1976). Since the temperature difference also changes the solvent concentration, it is important that the solvent have very low absorbance, and that its absorbance, if significant, have no fine structure so as to simplify the correction.

The relatively large thermal expansivity of many organic solvents must contribute signifi- cantly. Some idea of the magnitude of the effect can be seen in the TP spectrum of N- acetyltyrosine ethyl ester (ATEE) in methanol (Nicola & Leach, 1976). Comparedwith TPspec- tra in aqueous media the negative extremum in methanol is shifted about 2 nm to the red and is much reduced in magnitude, while the shorter wave length region (260-285nm) is much increased in magnitude. The 1% difference in volume for methanol produces for ATEE a AA of about 0.014, or 40% of the observed AAZm (Nicola & Leach, 1976), reduces I AA by a similar proportion, and shifts the extremum near 290 to the red. That is, when the magnitudes of overlapping extrema are altered, the positions of the extrema are also affected. A

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similar effect applies to spectra of chromophores in ethylene glycol. The thermally-induced concentration difference will be invoked in the next section on the TP spectrum of BSA.

Bovine serum albumin Leach & Smith (1972) presented the TP spectrum of BSA. The spectrum has a major negative extremum at 292-293 nm. The magnitude corresponds to 2.7 exposed tryptophyl residues, and the position to that of tryptophan models in water; but there is no negative extremum at about 288nm, typical of tyrosyl models in water. BSA contains two tryptophyl residues, both accessible to reaction with N-bromosuccinimide (Hurt & Leach, cited in (Leach & Smith, 1972; Nicola & Leach, 1976)). Leach & Smith (1972) and Nicola & Leach (1976), in a more detailed investigation and theoretical treatment, suggested that the 292-293 nm extremum arises from the two exposed tryptophyl residues, with the excess over two arising from tyrosyl side chains in regions of relatively low dielectric constant, which can be simulated by 6 0 4 5 % ethylene glycol.

We have measured the thermal perturbation of BSA under the same conditions as did Nicola and Leach, also using a Cary 14 spectro- photometer (0.1A slide wire). Our AE/AT at 292 nm is - 15 M-’ deg-’ cm-’ compared with -18 found by Nicola and Leach. Our value is so close to the AEIAT expected for two well- exposed tryptophyl residues that a search for tyrosyl residues would not be strongly indicated. Our A,, (278-279 nm) was 46 x lo3, close to that expected from the amjno acid composition. Therefore, our 20% lower value of A E ~ ~ / A T appears not to be the result of a 20% lower concentration. Because of the uncertainties involved in the variability of purity, conformation and instrument response, and because Herskovits & Sorensen (1968) found about seven exposed tyrosyls by means of solvent perturbation spectroscopy, we do not consider our result to be definitive. Therefore, we here examine the interpretation by Nicola and Leach of the thermal perturbation spectrum of BSA in water and glycol.

In the following discussion we suggest that there are other possible explanations of the Tp spectrum of BSA, but that there is no

unique explanation. The value of 2.7 exposed tryptophyl residues is based on a model compound value of /AT = 6.50 M-’ cm-’ deg-’ at pH7.4 (Table 2 , Nicola & Leach, 1976). Our value (Bello, 1970) is 6.67. Leach & Smith give a value (Table 2 , Leach & Smith, 1972) of 6.29 at pH5.57, but from their Fig.4 we calculate /AT - 7.1 . Thus the number of exposed tryptophyls may be under 2.5. (From Fig.5 of Nicola & Leach, 1976 we calculate a AeZw/AT of about 12.9. This is obviously the result of an error in the description of the experimental conditions given in the legend. The correct value is about one-half, presumably the 6.50 given in Table 2.) An experimental value 25% different from what the true value might be is not altogether bad, and might well be taken as an indication of there being few, if any, exposed tyrosyls. It is, therefore, a valuable contribution of Nicola and Leach to search for a tyrosyl contribution. An apparent exposure of 2.4-2.7 tryptophyl residues might arise from tryptophyl side chains in an environment in which the long wave length extremum has a larger Ae than in water, obviat- ing the need to postulate a tyrosyl environment resembling 6 0 4 5 % glycol. No such model chromophoresolvent system has yet been reported ; but it is not impossible. This would imply that no tyrosyls were being thermally perturbed, in apparent contradiction to the solvent perturbation data of Herskovits & Sorensen (1968) which showed the presence of about seven exposed tyrosyls. We shall return to this point later.

Nicola and Leach stated that the absence of a negative extremum at 280 and 288nm for BSA argues against a contribution from “normal” tyrosyl, and the absence of a negative extremum at 282 nm argues against a “simple tryptophan spectrum”. The absence of 280 and 288nm extrema could, naively, be taken as evidence of no exposure of tyrosyls. However, BSA does show a negative extremum at 281 nm (Fig. 10, Nicola & Leach (1976)), in the form of a high valley between the positive 278 and 285nm peaks. The large magnitude of the 285-270nm region (and the height of the valley at 281nm), in comparison with the tyrosyl and tryptophyl models in aqueous buffer, could, to a large extent, be accounted

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for by two factors, the thermal concentration difference and the effect of buried tyrosyls. For 21 tyrosyl residues E,, is about 34 x lo3. This is based on 1600 per tyrosyl, since buried residues have larger E values than exposed residues. If some tyrosyls are exposed a smaller base of calculation results; but the contribution from disulfides is also needed. Therefore, 34 x lo3 is reasonable value for rough estimation. A 0.27% concentration difference between 10' and 25' would give a AEIAT = 6M-' cm-' deg-'. The remaining positive region would arise from the true thermal perturbation of the two exposed tryptophyls and from the buried tyrosyls. The latter contribution may be estimated from the data of Nicola and Leach for acetyltyrosine ethyl ester (ATEE) in dry poly- vinyl alcohol films. For 21 buried tyrosyls AT = 8, which added to the concentration effect gives 14, about half of the observed value of 28 for BSA. These corrections of the BSA spectrum would bring the 281 nm valley close to zero according to Fig. 10 of Nicola and Leach (1976). We cannot be sure that the valley would still exist, since we do not know the exact shape of the corrections for the buried chromophores. Tyrosyl models in water and in dry poly(viny1 alcohol) films show a thermal perturbation extremum at about 280 nm (Nicola & Leach, 1976; Bello, 1969) and the expected value of AemlAT for six tyrosyls exposed to water is -2M" cm-' deg-' (calculated from Fig. 4, Nicola & Leach (1976)) and about -2 for 21 tyrosyls in the poly(viny1 alcohol) film (Fig. 8, Nicola & Leach (1976)). A negative extremum at 279-281 nm is not suitable for the estimation of exposed tyrosyls, because its small magnitude makes it susceptible to distorting influences, because aqueous and dry fdm environments cannot easily be differ- entiated, because tryptophan also has a negative extremum near this wave length, and because when exposed tyrosyl and tryptophyl are both present, the extremum at 280-281 largely vanishes through cancellation of positive and negative AE values (Fig. 1, Bello (1970)).

The correction for the thermal perturbation of buried groups requires a correction of its own arising from twodimensional contraction of the cold film (contraction normal to the film plane will have no effect). This will be smaller

than contraction of liquids generally. Thus, the correction for buried chromophores will be somewhat smaller in the positive region, but will be somewhat greater in the negative region.

Readers may note that Nicola and Leach, in their Fig. 11, show a BSA spectrum strongly at variance with that of Fig. 10, in that in Fig. 10 Ae2n/A~292 = 1.56 and in Fig. 11 A E ~ ~ ~ / A E ~ ~ = 0.72, although Ae2= is about the same in both Figures, that is, A~277 in Fig. 10 is about twice A ~ 2 7 7 in Fig. 11. The above corrections applied to Fig. 11 would largely eliminate the positive lobe. Nicola and Leach have noted elsewhere that Fig. 10 is the correct one. Our experiment under as closely similar conditions as possible confirms this, our = 1.57, in excellent agreement with that of Fig. 10. Our spectrum showed better resolution of the extrema at 255-270 nm, comparable to that of Fig. 11, and, as noted above, the magnitude of our spectrum was about 20% smaller. The calculated spectrum for BSA (lower part of Fig. 10) overestimates the possible contribution from the six tyrosyls postulated to be in a glycol-like environment. That is, Nicola and Leach use the spectrum of ATEE in 85% glycol, which contains a significant mismatch contribution arising from the greater coefficient of thermal expansion of glycol compared with water. Such a contribution would not be applicable to the hypothesized special tyrosyls of BSA in aqueous medium. This correction worsens the fit between experimental and calculated spectra.

Nicola and Leach contend that the correction for buried tyrosyls is essentially zero. In response to this we note the following. Fig. 8 of Nicola and Leach shows that the positive lobe of the thermal perturbation spectrum, from about 280 to 265 nm, is substantially indepen- dent of the extent of dehydration, and except for the wettest film, this is true up to 285nm. From these results there is no ground for expecting a completely dry film to be signifi- cantly different in this wave length range. Therefore our correction of 8 M" cm-' deg-' at 277 nm is in accord with these data. The long wave length region, around 290nm, shows a strong dependence on the mode of dehydration. The driest film was dehydrated for one week over P2 05. It appears unlikely that any water

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remaining could resemble mobile water; remaining water would be a small fraction of the total film mass, and would be strongly held, presumably bonded to polymer hydroxyl groups. It would, in effect, be a part of the polymer structure although it may be able to migrate. We may not be justified in considering this film as dry as the interior of a protein, but Nicola and Leach have not shown that further dehydration would reduce Ae at 290 nm to essentially zero. Ae is already only 17% that of ATEE in water; this is small, but the sum over numerous tyrosyls is significant. There may be little dehydration remaining to accomplish, and that little may produce little, if any, further change in Ae/AT. It may be that poly(viny1 alcohol) is not a valid model for the protein environment. However, Nicola and Leach presented the fdm data as part of the rationale for interpreting the TP spectrum of BSA, and our alternate interpretations are based on their data. A good fit between experiment and theory is not possible without a buried tyrosyl contribution to the positive region of the thermal perturbation spectrum.

Corrections must be made at the long wave length extremum. The corrections for the concentration difference will be made only for tyrosyl, since we assume that the tryptophyls are well exposed and, therefore, that the concentration difference for BSA will cancel that for the model compound. The correction for concentration difference is small, increasing 1 Ae2=/AT I by about 1 M" cm-' deg-' , but the correction for buried tyrosyls is larger, +8 M-' cm-' deg-' , making the net Ae2=/AT = -12, or two exposed tryptophyls depending somewhat on the choice of model value of Ae2=/AT. This is in good agreement with solvent perturbation (Herskovits & Sorensen, 1968) and N-bromosuccinimide data which indicate that two tryptophyls are exposed. Therefore, it may not be necessary to resort to the device of Nicola and Leach, namely, calculation of a TP spectrum based on Ac of six tyrosyls in a medium resembling 60-85% ethylene glycol. Again we note that this explanation is not in agreement with the indication by solvent perturbation that six-seven tyrosyls are exposed.

It is also possible that Ae2=/AT represents

one exposed tryptophyl and several tyrosyls. This interpretation is inconsistent with solvent perturbation data indicating exposure of two tryptophyl residues, but is consistent with the thermal perturbation spectrum of BSA in 60% glycol, to be discussed below.

Nicola and Leach discussed three types of support for their idea that part of the 292- 293nm extremum of BSA arises from about six tyrosyls exposed to a pauci-aqueous environment having the properties of solvent of lower dielectric constant. The first support was the calculation of a spectrum based on that of two normally exposed tryptophyls and of six tyrosyls in 85% ethylene glycol. Their calculated spectrum matched the observed spectrum well at the long wave length negative extremum (their Fig. lo), but not so well in the region of positive Ae. A correct calculation for curve fitting requires inclusion of the concentration difference (or its experimental elimination) and the inclusion of the contribution from buried groups, as indicated above.

The second support for the proposal of five-six tyrosyls in an environment similar to that of 60-85% glycol was the TP spectrum of BSA in 60% glycol (Fig. 11 of Nicola & Leach (1976)). The rationale was that the exposed tyrosyls, being already in a similar environment, would not be affected, but that the two exposed tryptophyls, being in an aqueous environment, would respond like model tryptophan in 60% glycol, with a red-shift, diminution of the long wave length extremum, and an increase in Ae in the region of positive A€. They observed, in fact, just these anticipated results. That is, the negative AA at 292nm shrank by 45%, a new negative extremum appeared at 303 nm, and the positive AA at 290-270 nm increased. That is, there was an increase compared with the incorrect TP spectrum of aqueous BSA shown in Fig. 11 ; there would be no increase over that shown in Fig. 10. (it is not clear why Fig. 11 for aqueous BSA differs from Fig. 10. Perhaps the 50% greater absorbance (2.7A) of the solution used for Fig. 11 was a factor.) About one-third of the increase in the 290- 270 region would arise from the concentration mismatch. Fortunately, Figs. 10 and 11 for BSA in water are in agreement at the 292nm extremum. "he concentration correction at the

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292nm extremum of BSA in 60% glycol in Fig. 11 would add 0.0012 AA to the observed AA of about 0.01. This corresponds to Ae/AT = - 12 M-' cm-' deg-' . Correcting for buried tyrosyls (from the dry film data) would decrease I Ae2a /AT I by -8 M-' cm" deg-' , leaving about -4 M-' cm-' deg-' , compared with a value of about -5 per tryptophyl in 60% glycol, obtained by interpolation in the data of Fig. 5 of Nicola and Leach. Alternatively,the corrected A E ~ /AT is equivalent to about two tyrosyls in 60-80% glycol (Fig. 4b, Nicola & h a c h (1976)). Considering the uncertainties in these corrections, the number of exposed tyrosyls indicated by for BSA in glycol might be as low as zero or in the range of five-six suggested by Nicola and Leach. From the spectrum alone it is not possible to tell if the 292 nm extremum arises from tyrosyl or tryptophyl.

The appearance of the 303nm extremum clearly shows that at least one tryptophyl is affected by 60% glycol. (A tyrosyl so far to the red is highly unlikely.) A E ~ ~ / A T for BSA in 60% glycol is -6M-' cm-' deg-'. But this should be corrected for the contribution of the toe of the 293nm extremum. A reasonable approximation to this correction makes Aem3 / AT about -4 to -4.5 M-' cm-' deg-' . Nicola and Leach do not give a spectrum for model tryptophan in 60% glycol, but they show that in 40% glycol AE/AT is -6.2 (the extremum being still at 292nm), and in 100% glycol Ae/AT=-3.8 (the extremum being at about 300nm). Thus, the 303nm extremum could represent one tryptophyl in a medium like 60% glycol (but red-shifted), while the remaining diminished extremum at 292 nm could represent the other tryptophyl residue. It is not clear why only one tryptophyl would so respond, while both respond to solvent perturbation with 20% glycol. It should be noted that the new 303 nm extremum is considerably more to the red of that expected for tryptophan in 60% glycol or even 100% glycol (compare Figs. 5 and 11 of Nicola & h a c h (1976)). If the 303nm extremum for BSA in 60% glycol arises from both tryptophyls (with the remaining 292nm extremum from tyrosyl), Aem/AT is about -2 to -2.5 M-' cm-' deg-' per tryptophyl, or about one-half of the model compound value. It is difficult to compare with model compound

data since the extremum for tryptophan in 60% glycol is estimated at about 296nm, and in 100% glycol is at 300 nm (Fig. 5,Nicola & Leach (1976)). If we are to place any value on model compound data, we must assign the 303nm extremum to one tryptophyl, and the 292nm extremum to the other or to a few tyrosyls, as discussed above.

An alternative explanation is that both tryptophyls contribute to both the 292 and 303 nm extrema of BSA in 60% glycol. We have observed that two solvents of quite different properties produce double extrema near these wave lengths with tryptophyl models. We have shown (Bello & Bello, 1973) that Lys-Trp-Lys in 0.03M sodium dodecyl sulfate gives two negative TP extrema, at 292 and 303 nm. These may represent two distinct environments. Extrema at both 292 and 303nm were also observed for N-AcTrp-NH2 in aqueous 16M potassium formate (Bello, 1970). Such an effect has not been observed for model chromophores in glycol, but a priori, cannot be ruled out for a protein in glycol.

Nicola and Leach proposed that the tyrosyls in the hypothesized relatively non-polar environment are in crevices in a motile region of the protein with partial access to water, the result being "similar to that of a model chromophore in a mixed solvent". Chromophores in a crevice may behave differently from the exposed chromophores of models. The crevice solvent may be highly restricted in its mobility, respon- ding little to temperature, with the result that crevice chromophores may behave like buried chromophores. Buried chromophores appear to be little affected by temperature, as had been suggested by us (Bello, 1969), and sup- ported by the work of Nicola and h a c h on chromophores in polymer films. (However, as we have shown above, although buried chromophores may have small AE values, the sum over many buried chromophores may be significant.) 'Ihe chromophores in such a crevice may be effectively buried as indicated by TP, but may be accessible to perturbing solvent and chemical reagents. Thus, TP spectra, on the one hand, and solvent perturbation spectra and chemical modification, on the other hand, may give quite different results for crevice chromophores.

The glycol TP experiment of Nicola and

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h a c h is based on a rationale inconsistent with the solvent perturbation data of Herskovits and Sorensen. It requires that the tyrosyls in the crevices in their interaction with a small amount of water shall give a thermal perturbation spectrum as though in 60-80% glycol, but when in contact with 60-80% glycol shall give an unchanged thermal perturbation spectrum. Nevertheless, they must give a normal solvent perturbation spectrum with 20% glycol. Our suggestion overcomes this difficulty, although it is, for now, speculative.

The third support for the proposal of Nicola and Leach (that five-six tyrosyls are in an exposed but special environment) was acetylation with N-acetylimidazole. They showed that about six tyrosyls of BSA react with N-acetylimidazole. The TP spectrum of Ac6BSA was mark- edly different from that of BSA, except at the 292 nm extremum, for which AeIAT = -16 M-' cm-' deg-' , equivalent to about 2.5 tryptophyl residues. The broad positive region at 295-260 for BSA was virtually absent, being replaced by negative Ae, except for a small positive extremum at about 278nm. The observed TP spectrum of Ac6BSA does not have the Ae/AT expected from the concentration mismatch and the 15 buried tyrosyl residues. For the 15 unacetylated tyrosyls and two tryptophyls E~~ must be about 35 x lo3 (not counting disulfides), and the temperature difference of 15' must produce a concentration difference with Ae2,/AT of about 6 M-' cm-' deg-' in the 275-285 nm region. In addition, the buried tyrosyls should produce a Ae of about 7 M-' cm-' deg-' (as calculated above from dry film data). The sum of these corrections is about 13 M-' cm-' deg-' , which is almost double the largest observed Ae/AT of Fig. 12, (Nicola & Leach, 1976), for Ac6BSA. That is, Ae/AT is only one-third of the expected value. The buried chromophore effect in the protein interior is not necessarily similar to that in poly(viny1 alcohol), and could, as far as we now know, result in a negative Ae in part or all of the 278-285 nm region, giving rise to the observed TP spectrum of Ac6BSA. But the TP spectrum of native BSA (Fig. 10, Nicola & Leach (1976)) appears to require a positive Ae from buried groups. This caveat about the model for buried tyrosyl applies to all the fore-

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going calculations. It will be necessary to study other model solid environments.

Further, from the data of Figs. 4b and 6 (Nicola & Leach, 1976) the acylation of six tyrosyls in an 80% glycol-like medium should decrease Ae2= /AT by about 7 M-' cm-' deg-' . The actual reduction from that shown in Fig. 10 is 2 M-' cm-' deg-' , which is within experimental uncertainty.

In addition, the TP spectrum of Ac6BSA disagrees with expectation in another way. The value of AelAT at 275 nm for a tyrosyl in 80% glycol is 2.2 M-' cm-' deg-' , of which about 0.5 arises from the concentration mismatch. An 0-acetyltyrosyl residue in 80% glycol would be expected to have a AE/AT 5 -0.5 (compared with -0.8 in water). Therefore, acetylation of a tyrosyl in a crevice having the properties suggested by Nicola and Leach would result in a decrease of at most 2.7 M-' cm-' deg-' , more probably 2.2 with deletion of the concentration mismatch. For six tyrosyls, acetylation would decrease Ae/AT by 13 (at most 16). From Figs. 10 and 12, Ae/AT decreased by 28M-' cm-' deg-' at 275nm, or about twice the expected value. These factors make it difficult to under- stand the meaning of the data.

(Nicola & Leach (1976) showed TP spectra of N,O-diacetyltyrosine. We (Bello, 1969) had earlier shown such spectra. More recently we have found that our spectra contained significant errors because of the presence of some unacetylated phenolic groups. Our newer results agree well with those of Nicola and Leach, but with a smaller about -0.1 M-' cm-' deg-' instead of about -0.2.)

These considerations indicate that the interpretation of TP spectra is complex, particu- larly when there are numerous buried groups contributing to the spectrum. Other methods of studying the chromophores of BSA, in particular fluorescence of the tryptophyl residues, have led to conflicting interpretations as to solvation, location in the surface or crevices and as to whether the two tryptophyls are in similar environments (Luk, 1971; Holmes & Robbins, 1974; Feldman et al., 1975).

Nicola and Leach estimated, from Ae/AT at 268 nm, that 22 phenylalanine residues are exposed. They recognized that the super- position of the spectra of the other chromo-


phores must result in significant error, but they suggested that a large fraction of the phenyl- alanyl residues is exposed. This calculation is not valid because AeZa/AT for BSA is positive, and Ae/AT for phenylalanine is negative throughout the wave length range considered (Plttz & Bello, 1970; Bello & Bello, 1976).

Nicola and Leach have informed us that the correct calculation would use the difference between the observed spectrum and the calculated spectrum (calculated without a phenylalanine contribution). From Fig. 10 (Nicola & Leach, 1976) this would result in a Aez,/AT = -8 M-' cm-' deg-' , equivalent to 16 exposed phenylalanine residues. Our AE/AT for phenylalanine is -0.57 at 268nm (Bello & Eello, 1976), which gives 14 exposed residues in BSA. There may be evidence in support of the exposure of phenylalanines in the thermal perturbation of Ac6BSA in their Fig. 12. The negative extrema between 250 and 260 nm suggest the presence of exposed phenylalanine (Plttz & Bello, 1970). However, 0-acetyltyrosine gives extrema at about the same wave lengths and of similar magnitude. As Nicola and Leach correctly point out, an estimate for phenylalanine may be strongly distorted by the cumulative uncertainties in the summation of the contributions of other chromophores. The possibility of a considerable number of exposed phenylalanines is an intriguing idea considering the hydrophobic character of th is side chain.

pH 8.1, and alkylation with the stoichiometric quantity of bromoacetic acid at pH 8 2, followed by filtration through Sephadex G-25 and freeze- drying. The amino acid analysis for RCM- lysozyme was normal and showed 7.7 residues (theory 8) of carboxymethylcysteine.

Thermal perturbation experiments were done as described earlier (Bello, 1970). In this and all earlier TP work water at 26' was circu- lated through the cell compartment walls.

REFERENCES

Bello, J. (1969) Biochemistry 8,4542-4550 Bello, J. (1970) Biochemistry 9,3563-3568 Bello, J. & Bello, H. R. (1973) Eur. J. Biochem. 34,

Bello, J. & Bello, H. R. (1976) Arch. Eiochem. Eio-

Canfield, R. E. (1963) J. EioJ. Chem. 238, 2698-

Feldman, I., Young, D. & McGuire, R. (1975) Blo-

Herskovits, T. T. & Sorensen, M., Sr. (1968) Eiochem-

Holmes, L. G. & Robbins, F. M. (1974) Photochem.

Leach, S. J . 81. Smith, J. A. (1972)Int. J. hot. Res. 4 ,

Luk, C . K. (1971) Biopolymers 10,229--241 Nicola, N. A. & Leach, S. J. (1976) Int. J. Pept. h t .

Pittz, E . P. & Bello, J. (1970) Trans. Faraday Soc. 66,

5 35 -5 38

PhyS. 172,608-610

2707

polymers 14,335-351

istry 7,2533-2542

Photobid 19,361-366

11-19

Res. 8,393-415

5 37-545

EXPERIMENTAL PROCEDURES Address: Jake Eello

RCAM-lysozyme was prepared as described Department of Biophysics earlier (Bello, 1970). RCM-lysozyme- was pre- Rowell park Memorial Institute pared by reduction with a 15-fold ratio of 6 6 6 a m Street dithiothreitol to protein disulfides in 8 M Buffalo, New york 14263 urea (freshly deionized), 0.1 M Tris-acetate, u.S.A.

79

interpretation of thermal perturbation spectra of proteins

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