Alkaline denaturation of bovine serum albumin. II. Analysis by isoelectric focusing in polyacrylamide gels.

Abstract

Bovine serum albumins exposed to pH 11.9 for various periods (5min.-6hr.) at 20°C were analyzed by isoelectric focusing in polyacrylamide gels. Native BSA** monomers gave, in the electrofocusing pattern, three peaks with pI values 4.84, 5.15, and 5.39. After exposure to alkali, apparent shifts in the isoelectric point of some of the peaks and the appearance of new peaks with pI values 5.48, 5. 52, 5.70, 5.85, and 5.91 were observed.Components of these peaks are broadly divisible into the following three fractions: 1. The“first fraction”which consists of the main components of native BSA monomer (pI≤5.15). 2. The “second fraction”composed of SS-interchanged isomers (5.15 5.70). 3. The“third fraction” consisting of irreversibly unfolded products (pI≥5.70). The alkaline denaturation can be expressed by the following reaction diagram: the“first fraction”→the“second fraction”→the“third fraction”.When the solution of native BSA monomer is exposed to pH 11.9 at 20°C for 4hr., new components 1, 1″, 1′, 2, and 3 are formed (the“second reaction”). Each of these components was isolated in the pure state by preparative disc gel electrophoresis and analyzed by electrofocusing. The results obtained are as follows: The component 1 and the native BSA monomer are composed of only the “first and second fractions”, but differ from each other in the situation of ionizable groups. Components 1″, 1′, 2, and 3 each consists of all the three“fractions”. The components 2 and 3 are similar and 1″and 1 also resemble each other, both in the state of charged groups. It is postulated that in the“second reaction”fatty acids, the impurity bound to BSA, migrate from the component 1″ to the component 1 in the course of the reaction, 1→1″.