Modification of Protein Properties by Change in Charge

Abstract

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...

Concanavalin A Derivatives with Altered Biological Activities

“…The rapid rise in extent of coupling after dialysis and lyophilization probably involves conformational changes in the protein due to reversible dissociation of succinyl groups during dialysis or to lyophilization, rather than to the mere addition of fresh reagent. A similar effect has been reported for succinylation of chymotrypsinogen (21).…”

Molecular thermodynamics of aqueous two‐phase systems for bioseparations

“…Tables 3 and 4 present the phase-diagram data for the two two-phase systems studied. The proteins studied here are well characterized with respect to surface charge as a function of pH (Tanford and Wagner, 1954;Tanford et al, 1955;Sakakibara and Hamaguchi, 1968;Shiao et al, 1972;Horn and Heuck, 1983). As stated above, chlorides and sulfates added to the phase systems do not alter the pH of the phases significantly from neutral pH; however, the pH of 50 mM potassium phosphate is 5.5.…”

A molecular-thermodynamic model for the interactions between globular proteins in aqueous solutions: Applications to bovine serum albumin (BSA), lysozyme, α-chymotrypsin, and immuno-gamma-globulins (IgG) solutions

“…The dispersion energy parameter ε 1 /k B for α-chymotrypsin is regressed from the osmotic pressure data of Haynes et al [2], and has a value of 466.32 K. We regard the molecular weight and dispersion energy parameter ε 1 /k B for a specific protein as constants when pH and ionic strength in solutions are changed. The used net charge of α-chymotrypsin molecule is the titration data of Marini and Wunsch [22], and Shiao et al [23], which can be found in Table 2.…”