A flow microcalorimeter has been employed to measure the enthalpies of binding of indole, N-acetybtryptophan, and proflavin to a-chymotrypsin at pH 7.8 and also the heats of dilution of a-chymotrypsin and proflavin at the same pH. The following points are discussed. (1) The studies of the heats of dilution of a-chymotrypsin as a function of enzyme concentration lead to the suggestion that monomer-dimer equilibrium of this enzyme exists at pH 7.8. The calculated thermodynamic functions for this equilibrium are consistent with the view that the dimeric forms of the enzyme can be identified as the enzyme-substrate intermediates preceding the autolysis reaction. (2) The heat effects due to the dilution of proflavin are shown to be consistent with the assumption that the self-association of this compound can be T he binding of various inhibitors to a-CT1 has been extensively investigated by various techniques. Niemann and his colleagues examined over 100 compounds, determining their inhibition constants toward the CT-catalyzed hydrolysis of acetyl-L-valine (Hein and Niemann, 1962;Wallace et al., 1963). From those studies, useful information concerning the topography of the active site of a-CT was obtained. The comparison of binding constants of some charged and uncharged inhibitors as a function of pH (Foster and Niemann, 1955;Johnson and Knowles, 1966) has led to the suggestion that the active site of a-CT is negatively charged at neutral pH values where the molecule is enzymically active. This suggestion has recently received support from X-ray studies of a-CT (Blow et al., 1969).The binding of various substrates and inhibitors to a-CT produces an enhancement of the fluorescence of the enzyme (Sturtevant, 1962). The fluorescence increase takes place with first-order rate constants of about 1 sec-1 for widely different ligands, the rate constants being independent of ligand concentrations. These findings suggest that conformational changes in a-CT, which are included by the binding of substrates and inhibitors, are responsible for the observed * From the adequately represented by a series of equilibria involving dimer, trimer, and other higher polymers. The large negative enthalpy of polymerization (-4.0 kcal/mole) shows that the self-association of proflavin is accompanied by an unfavorable entropy change.(3) The apparent heats of binding of inhibitors to a-chymotrypsin were observed to be strongly dependent on enzyme concentration. These observations lead to the suggestion that the dimeric forms of the enzyme are incapable of binding inhibitors. (4) The enthalpies of binding of indole, N-acetyl-D-tryptophan, and proflavin to a-chymotrypsin were observed to be -15.2, -19.0, and -11.3 kcal/ mole, respectively, at pH 7.8. On the basis of these numbers it is concluded that conformational changes in the enzyme are induced by the binding of inhibitors. changes in fluorescence emission. As an aid to understanding the nature of the conformational changes mentioned above and also the various forces involved in the binding r...

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SynopsisThe heats of binding of protons m b to three globular proteins, lysozyme, chymotrysinogen A, and oxidized cytochrome c, from pH 11-2 or lower a t 25°C were determined by flow microcalorimetry. In addition, the acid-base titrations of cbymotrypsinogen A and oxidized cytochrome c were investigated under conditions similar to those used in the calorimetric experiments. The results of the calorimetric experiments and the acid-base titrations for lysozyme and chymotrypsinogen A in the neutral and alkaline pH regions are in accord with the Linderstrom-Lang model with a pH-independent electrostatic factor. However, in order to interpret the results on oxidized cytochrome c in the same pH region, it is necessary to assume the protonation of two unidentified groups; one of these groups, with AHb = -18 kcal/ mol and pK, = 9.4, was detected in previous work [Watt, G . D. & Sturtevant, J. M. (1969) Biochemistry 8,45671. The normal heats of protonation for carboxyl, c-amino, phenolic, a-amino, and imidazole groups on globular proteins as deduced from the study are 0, -10.5, -6.3, -10.0, and -6.3 kcal/mol, respectively. AHb of chymotrypsinogen A between pH 4.5 and 1.3 is +30 kcal/mol. This value, which is undoubtedly too large to be accounted for by the protonation of normal carboxyl groups, leads to the conclusion that this protein undergoes a pH-induced conformational change in this pH region. The same idea can be applied to explain the abnormal AHb observed for oxidized cytochrome c in the alkaline pH region.

synopsisThermal denaturation of calf thymus DNA at both alkaline and neutral pH values was studied by differential scanning calorimetry. It was shown that the dependence of the enthalpy of transition on pH and salt concentration could be accounted for on the basis of a heat capacity change of +40 cal deg-1 (base pair)-'. In the pH range between 10.3 and 11.3, a release of 0.6 proton per base pair was calculated from the pH dependence of the melting temperature. The heat effect associated with the release of this proton was calculated to be 5 kcal mole-'.

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...