SynopsisThis article presents evidence for the existence of a specific h e a r relationship between the entropy change and the enthalpy change in a variety of processes of small solutes in water solution. The processes include solvation of ions and nonelectrolytes, hydrolysis, oxidation-reduction, ionization of weak electrolytes, and quenching of indole fluorescence among others. The values of the proportionality constant, called the compensation temperature, lie in a relatively narrow range, from about 250 to 315"K, for all these processes. Such behavior can be a consequence of experimental errors but for a number of the processes the precision of the data is sufficient, to show that the enthalpy-entropy compensation pattern is real. It is tentatively concluded that the pat-tern is real, very cornmon and a consequence of the properties of liquid water as a solvent regardless of the solutes and the solute processes studied. As such the phenomenon requires that theoretical treatments of solute processes in water be expanded by inclusion of a specific treatment of the characteristic of water responsible for compensation behavior. The possible bases of the effect are proposed to be temperatureindependent heat-capacity changes and/or shifts in concentrations of the two phenomenologically significant species of water. The relationship of these alternatives to the two-state process of water suggested by spectroscopic and relaxation studies is examined. The existence of a similar and probably identical relationship between enthalpy and entropy change in a variety of protein reactions suggests that liquid water plays a direct role in many protein processes and may be a common participant in the physiological function of prot.eins. It is proposed that the linear enthalpy-entropy relationship be used as a diagnostic test for the participation of water in protein processes. On t,his basis the catalytic processes of chyniotrypsin and acetylcholinesterase are dominated by the properties of bulk wat,er. The binding of oxygen by hemoglobin may fall in the same category. Similarities and difyerences in the behavior of small-solute and protein processes are examined to show how they may be related. No positive conclusions are established, but it is possible that protein processes are coupled to water via expansions and contractions of the protein and that in general the special pattern of enthalpy-entropy compensation is a consequence of the properties of water which require that expansions and contractions of solut,es efiect. changes in the free volume of t.he nearby liquid water. It is shown that proteins can be expect,ed t,o respond lo changes in nearby water and interfacial free energy by expailsions arid cont.ract.ions. Such responses may explain a variety of currently unexplained cliitracteristics of protein solutions. More generally, t,he enthalpy-entropy conipeiisation pattern appears to be the thermodynamic manifestation of "st,ructrira making" and "structure breaking," operationally defined t,erms much used in discussio...

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The stepwise synthesis of peptides by the initial attachment of an amino acid /-butyloxycarbonylhydrazide through its -amino group to a polystyrene resin was investigated. The resin-amino acid azide was generated quantitatively and coupled with another amino acid r-butyloxycarbonylhydrazide. The peptide chain was elongated by further azi^e couplings. Finally, the C-terminal amino acid was added as a r-butyl ester.

Succinylated derivatives of bovine chymotrypsinogen A and n-chymotrypsin were prepared by treatment of the native proteins with succinic anhydride; 60°/, of the 6-amino groups of the 13 lysine residues form stable succinylated products. The large negative charge a t neutral pH is reflected in electrophoretic and titration behavior but absorption spectra, rotatory-dispersion spectra and reversible unfolding behavior indicate that the succinylated species are conformationally similar to the parent proteins. Succinylated chymotrypsin is active as an esterase with N-acetyl-L-tryptophan ethyl ester with a small decrease in the maximum-velocity parameter. The p H dependence suggests that three ionizing groups influence catalytic behavior. The apparent p K , values analyzed in terms of three groups are shifted relative to n-chymotrypsin. Thermal-unfolding studies of succinylated chymotrypsinogen demonstrate the presence of substates near p H 7 but is consistent with two-state behavior a t other p H values.In order to study the role of protein charge on the folded stability, conformational dynamics, hydrogen-exchange behavior and entropy-enthalpy compensation pattern of the proteins of the chymotrypsinogen A family, it is necessary to prepare and characterize a varienty of members of this family, chemically modifled to have wide variations in charge in the p H range from 6 to 9.5. Succinylation has been widely used to produce moditications of this type to proteins such as subtilopeptidase The reaction of succinic anhydride with proteins a t neutral pH in water solution adds the group -COCH,CH,COOto free amino groups. It also reacts with phenol, imidazole, serine hydroxyl and threonine hydroxyl groups but on prolonged dialysis against water, the product hydrolyzes to restore the original groups [i]. I n this paper we report the results obtained in characterizing succinylated chymotrypsinogen A, and a-chymotrypsin at neutral pH. It is hoped that the basic information provided in this report will promote further study of these derivatives. EXPERIMENTAL PROCEDURE MaterialsThree-times crystallized chymotrypsinogen and chymotrypsin were purchased from Worthington Abbreviation. ORD, optical-rotatory dispersion.Biochemical Corp. and used without further purification. Reagent grade succinic a.nhydride was obtained from Eastman Kodak Co. Method of PreprationOne gram of chymotrypsinogen was dissolved in 50ml 0.05M phosphate buffer pH 7.5 a t room temperature; 50 mg solid succinic anhydride was slowly added to the protein solution. I n order to maintain the pH of the solution at 7.5, I N NaOH was added continuously. The addition of the total amount of succinic anhydride usually took about 30min. The reaction was carried out for another 10 min and then stopped by adjusting the pH of the solution to 4.5 with I N HCI. The reaction mixture was exhaustively dialyzed against distilled water and then lyophilized. For reasons described below, the above procedure was repeated twice with the lyophilized sample as the starting material. I n o...

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