VOL. 8, NO. 5, MAY 1969 Perturbation of Proton and Detergent Binding Sites in Bovine Serum Albumin by Acetimidation" Joseph Avruch,t Jacqueline A. Reynolds,$ and John H. Reynolds8 ABSTRACT: Modification of bovine serum albumin by blocking the 57 lysine residues with methyl acetimidate hydrochloride leads to a protein with identical circular dichroism spectra with that of the native macromolecule but a slightly increased intrinsic viscosity. The binding isotherm of sodium dodecyl sulfate to both protein T he lysine residues of bovine serum albumin have been implicated in stabilization of the native state through charge interaction with the 40 buried carbox- ylate ions (Vijai and Foster, 1967; Foster and Clark, 1962) and in the binding sites on bovine serum albumin for sodium dodecyl sulfate (Markus zyxwvutsrq et zyxwvutsrq at., 1964). The last-named authors showed that sodium dodecyl sulfate stabilized native bovine serum albumin against urea denaturation but did not stabilize a modified form of bovine serum albumin in which the lysines were blocked to give an uncharged side chain. Tyrosine and tryp- tophan also have been shown to be involved in deter- gent binding to bovine serum albumin by Ray et at. (1966), Polet and Steinhardt (1968), and Reynolds et zyxwvutsrq al. (1967) who showed spectral shifts in these aromatic res- idues as the result of binding C&la sulfate and sul- fonate half-esters and C8-C12carboxylic acids to bovine serum albumin. The object of the present work was to block all ly- sine residues of bovine serum albumin with methyl acet- imidate hydrochloride (Habeeb, 1966; Wofsy and Singer, 1963)to form a modified protein with structure NH2+ PwNHZ+ zyxwvutsrq + PwNHCCH, I/ In so doing, the positive charge is left on all modified lysine residues but a longer side chain has been inserted. If €-amino groups are involved in charge-charge inter- actions with carboxylate groups and are also part of a set of binding sites for detergent ligands, it is reasonable to expect an alteration in both detergent and proton * From the Department of Microbiology, Washington University School of Medicine, St. Louis, Missouri 63 110, and Research Center, Monsanto Company, zyxwvutsrq St. Louis, Missouri 63141. ReceivedJanuary 6, 1969. t Present address: Department of Internal Medicine, Wash- ington University Medical School. 3 Department of Microbiology, Washington University Medical School. $ Research Center, Monsanto Co.: to whom reprint requests should be directed. species is identical. However, the binding-induced difference spectra are reduced in magnitude when the ligand interacts with the modified protein. Proton binding studies indicate a larger number of carboxylate and tyrosine groups are exposed in acetimidated bovine serum albumin than in native bovine serum albumin. binding properties of the modified protein. Previous studies of a limited nature (Habeeb, 1966) showed a small increase in the frictional ratio (<3 %)due to acet- imidation of bovine serum albumin. In this paper the hydrodynamic and optical properties of completely acetimidated bovine serum albumin are presented to show the extent of alteration of the native protein con- formation due to modification. The role of lysine resi- dues in binding of dodecyl sulfate and protons to the protein has been investigated. Methods Circular dichroism and optical rotatory dispersion were determined on a Cary 60 recording spectropolar- imeter using 1- and 0.1-cm path-length cells and appro- priate protein concentrations to maintain the dynode voltage below 4 zyxwvu kV. The slit was programmed to a con- stant 15-A band width. Ultraviolet difference spectra were measured in 1-cm tandem cells on a Cary 14 spectrophotometer. In all experiments reagents added to the sample cell were com- pensated in the reference beam. Sedimentation velocity was determined in a Spinco Model E ultracentrifuge at a rotor speed of 52,640 rpm. The ultracentrifuge was equipped with a schlieren op- tical system and photographs were taken at 8 min inter- vals after reaching constant speed. Viscosity measurements were made in Cannon-Man- ning semimicroviscometers in a thermostated oil bath held to +0.005". Flow times ranged from 250 to 400 sec and were reproducible to 1 0 . 2 sec. Equilibrium dialysis techniques for measuring the binding of sodium dodecyl sulfate to bovine serum albumin as well as the spectrophotometric method of analysis for free detergent by extraction of a methylene blue-dodecyl sulfate complex into chloro- form have been described previously (Ray et at., 1966; Reynolds et at., 1967). All experiments were carried out at 2" in 0.033 ionic strength phosphate buffer (pH 5.6). Hydrogen ion titration curves were obtained by con- tinuous titration with an International Instrument Co. 1855 PERTURBATION OF BINDING SITES IN BOVINE SERUM ALBUMIN