Analysis of Homozygous Serum α1-Antitrypsins: Effects of Neuraminidase

Abstract

Extract: The molecular basis for the complex electrophoretic banding patterns of serum α1-antitrypsins of different protease inhibitor (PI) types is unknown. In order to demonstrate the role of sialic acid residues in this polymorphism, serums of homozygous PI, types FF, MM, and ZZ, were studied by acid starch and crossed electrophoresis, before and after treatment with neuraminidase. Increasing enzyme concentrations, temperatures, and times of incubation led to greater release of sialic acid and progressive slowing of PI bands. Exhaustive incubation of the three serum types with neuraminidase produced a single broad cathodal peak of α1-antitrypsin. Isoelectric focusing in gel with a pH 4–6 gradient, followed by immunoprecipitation of α1-antitrypsin bands, resulted in complex banding patterns with the expected differences in isoelectric points among the three serum types. After exhaustive neuraminidase treatment, each of the banding patterns remained complex, but shifted toward a more basic pH. Although sialic acid residues are one of the major determinants of the net charge and electrophoretic mobility of different PI types, differences remain after removal of sialic acids which may represent other genetically determined differences in primary structure. Speculation: Differences in isoelectric point of the banding patterns of PI, FF, MM, and ZZ α1 -antitrypsins, remaining after removal of their terminal sialic acid residues, suggest that other primary molecular differences exist among these inherited variants. By analogy with other well known protein polymorphisms, these differences are likely to consist of variations in primary amino acid sequence. Detailed analysis of purified PI system variants will be required to establish this possibility.