Demonstation of human prealbumin by double one‐dimensional slab gel electrophoresis

Abstract

Abstract A double one‐dimensional (D 1‐D) electrophoretic method for slab gels with polyacrylamide gel electrophoresis (PAGE) followed by polyacrylamide gel isoelectric focusing (PAGIF) is described for the demonstration of prealbumin (PA) from human sera. Almost pure PA is obtained when serum is submitted to Page i an anionic discontinuous (glycine‐chloride) buffer system at alkaline pH. After page the Bromophenol Blue‐stained tracking dye boundary in the gel containing PA is cut out and the gel strips are overlayed on an isoelectric focusing gel. A final PAGIF pattern of PA is obtained which is hidden by other serum proteins when one‐dimensional PAGIF is performed. PAGIF of PA in a gel containing a gradient from 0 to 8 M urea perpendicular to the pH axis reveals the conversion of at least 7 diffuse bands with isoelectric point (pI) values between 4.2 and 4.7 into three sharp zones with pI values of 5.45 for one minor zone and 5.7 for the two other zones lying very close together. Among 1900 sera from adult pregnant women, 2 were found containing a genetically determined PA variant with a pI of 5.9 for a minor and a pI of 6.35 for a more intense zone in addition to the usual pattern. From the variant PAGE and PAGIF patterns and their distribution in the families of the carriers, it is concluded that PA is a tetramer under control of an autosomal structural gene which remains stable during PAGE abut dissociates into the monomers during PAGIF in the presence or presence of urea it is followed that PA under physiological conditions possibly forms complexes with more components than expected from its known binding capacity for thyroxine and retinal binding protein (RBP). This highly resolving K 1‐D electrophoretic technique allows the simultaneous analysis of up to 96 samples under identical experimental conditions. It is concluded that PA may be a good candidate for human mutation monitoring at the protein level.