Int. J . Peptide Protein Res. 10, 1977, 182-183 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the authot(s)

REPLY TO NICOLA A N D LEACH

J A K E B E L L 0

Received 29 March, accepted for publication 5 April 1977

Nicola and Leach have presented a rebuttal to some of the major points of my paper, In- terpretation of thermal perturbation spectra of proteins. They are correct in stating that the concentration correction is valid only for buried chromophores, since the exposed chromophores and models will be similarly affected. In our application of the concentration mismatch correction to the BSA spectrum of Nicola and Leach we used the entire chromophore content because we wished to show that the concen- tration mismatch affects the 280 nm extremum referred to by Nicola and Leach, in addition to indicating its effect on the magnitude of the extrema.

In connection with the TP spectrum of BSA shown in their Fig. 10 Nicola and Leach suggest a mismatch correction of 4.6 M-' cm-' deg-' , based on 15 buried tyrosyls. Ours was based on 21 tyrosyls and two tryptophyls. The use of 15 tyrosyls assumes, of course, that six are ex- posed. This is one of the questions at issue. Our interpretation of the data suggests that fewer than six tyrosyls, perhaps none, are exposed. If none are exposed, i.e., 21 are buried, the con- centration mismatch results in A€ = 6.4 M-' cm-' deg-'. The difference between 4.6 and 6.4 is a minor factor. Nicola and Leach calcu- late a correction of about 1 M-' cm-' deg-' at 290 nm, about equal to ours.

Nicola and Leach disagree with our correc- tions for buried chromophores. We had used the data of their Fig. 8, for ATEE in poly(viny1 alcohol) films, in particular the spectrum obtained with the driest film. They consider buried chromophores to have essentially zero thermal perturbation, and that this conclusion

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would be supported if A€ were plotted as a function of hydration and extrapolated to zero hydration. This is an untested hypothesis. Fig. 8 of Nicola and Leach shows that the positive lobe of the ATEE-film spectrum from 280 to 265 nm is substantially independent of the extent of hydration and, except for the wettest film, up to 285nm. From these results there is no ground for expecting a completely dry film to be significantly different. 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 1 week over P z 0 5 . We may not be justified in considering this film to be 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. There may be little dehydration remaining to be accomplished, and that may produce little, if any, further change in Ae/AT. Nicola and Leach presented film data as part of the ration- ale for interpreting the TP spectrum of BSA, and our alternate interpretations are based in part on their data. In essence, Nicola and Leach state that the calculation should not be based on the experiments they presented, but on an hypothesis as to the result of a complete drying experiment they did not do. However, they may actually have done the experiment (1 week with P z 0 5 ) and obtained a result different from their theory.

Nicola and Leach state that our interpret- ations of few or no exposed tyrosyls appeared to be inconsistent with solvent perturbation. However, we proposed a mode of reconciliation,

REPLY TO NICOLA AND LEACH

namely that tyrosine in a crevice, in contact with few water molecules, might behave as .buried toward thermal perturbation but as exposed toward solvent perturbation and chemical reagents. Nicola and Leach doubt this idea, stating that “It is hard to see how a chro- mophore environment so immobilized and shielded could protect the chromophore from contact with small water molecules in ‘the bulk solvent or from experiencing thermal changes in these interactions and yet allow access to bulkier solvent and chemical reagents”. -We did not state that the chromophores are “shielded”. Our idea is that the motion within a ‘crevice (which communicates with the bulk solvent) is very restricted, resulting in a thermal perturbation effect similar to that of buried chromophore, but still accessible to solvent perturbation or chemical reagents. Evidence for this is not available.

In their rebuttal Nicola and Leach amplify their idea as to the effect of glycol in a crevice, suggesting that glycol displaces apolar side chains from the vicinity of the tyrosine, and forming Type I hydrogen bonds. Since the nature of the interaction with glycol is not known, it is not clear that the proposal of Nicola and Leach should produce a solvent perturbation effect equivalent to tyrosyl in 6ulk 20% glycol. Glycol selectively bound in a crevice might produce a solvent perturbation effect quite different from that produced by mobile bulk 20% glycol. The fact that 20% glycol and chemical modification by N-acetyl- imidazole agree in indicating about 6-7 access- ible tyrosyls supports the idea that glycol is not selectively bound. Obviously, at this time both proposals are in the realm of speculation.

Nicola and Leach discuss the data in relation to TP in 60-85% glycol and solvent pertur- bation using 20% glycol. They state that “part of the difference of opinion arises from the fact ‘that Bell0 makes corrections for buried residues ,which.. . . .are not appropriate”. Since we have already discussed the film data, we do not repeat the arguments here.

Nicola and Leach note that we commented on the long wave length extremum in 60% glycol being too far to the red, that they did not, in their paper, make too much of this point. But one must not ignore model com-

pound data when a particular interpretation of protein data means that AE for a chromophore in the protein is taken as only one-half of that of the model.

Nicola and Leach suggest that the 303nm extremum for BSA in glycol might arise from a conformational change. If conformational changes are to be invoked, the explanation of the spectrum of BSA in glycol in terms of tyrosyls would be untenable. The confor- mational change we suggested for RCAM- lysozyme appears reasonable, because of the shape of the spectrum, namely, the large positive 293nm extremum. If there is a trans- conformation with BSA, its spectroscopic result is more subtle.

With regard to Ac6BSA, Nicola and Leach state that we used erroneous corrections, that the correction should be 4.6 M-’ cm-’ deg-’ at 278 nm, not our value of 14 M-’ cm-’ deg-’ . Their criticism is presumably based on, 1) our use of all tyrosyl and tryptophyl chromophores in the concentration mismatch correction, and 2) our correction for buried chromophores derived from the film data of their Fig. 8. Since in the case of Ac6BSA our doubt dealt with the absolute value of AE/AT, it is necessary to include all chromophores (buried as well as exposed) in the mismatch correction. The buried chromophore (film data) correction has been discussed above. Nicola and Leach may be correct about the 260-270 nm regions. Restric- ting our discussion to the region above 270 nm does not affect our proposals. Also, Nicola and Leach have not addressed themselves to our calculation that the decrease in AEIAT at 292 for Ac6 BSA is considerably smaller than expected for the acetylation of six tyrosyls in a glycol-like environment (-2 observed, versus -7 expected). The observed small decrease is at the border of experimental error and, if real, is in better accord with the reduction expected from the acetylation of six buried groups, based on the driest film data. Our paper also notes that the positive lobe of the BSA spec- trum is reduced on acetylation by about twice as much as expected for acetylation of six tyrosines. DR. JAKE BELL0 Roswell Park Memorial Institute 666 Elm Street Buffalo, New York 14263 U S A .

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