Albumin contamination of a purified human alpha 1-antitrypsin preparation does not affect either structural conformation or the electrophoretic mobility of the inhibitor.

A partially purified preparation of human alpha 1-antitrypsin (alpha 1-AT) shown to be 60% active as an inhibitor of bovine trypsin, was chosen as starting material to investigate the nature and extent of contamination by human serum albumin (HSA) and to see whether or not such a contamination was responsible for both the reduced inhibitory activity and the slower migratory rate of the proteinase inhibitor in SDS-PAGE. Immunoblotting analysis revealed the presence of HSA in the unprocessed preparation of alpha 1-AT which, both in denaturing and non-denaturing PAGEs, had the same mobility as HSA, appearing as a single band of 65 kDa. By submitting the unprocessed alpha 1-AT preparation to affinity chromatography on an Affi-Gel Blue chromatography column, an apparently highly purified and homogeneous form of alpha 1-AT was obtained, as confirmed by measurement of inhibitory activity and densitometric scanning of SDS-PAGE in non-reducing conditions. However, immunoblotting analysis still revealed the presence of HSA in the most active fractions of the inhibitor eluted from the column, and regardless of purification degree, the molecular mass of the inhibitor was always 65 kDa. Treatment with beta-mercaptoethanol led to separation in SDS-PAGE of HSA as a distinct band of about 10 kDa higher than the alpha 1-AT band, which instead maintained the same mobility as in non-reducing conditions. The results indicate that HSA has not been completely removed from alpha 1-AT, and its presence does not affect the electrophoretic mobility of the inhibitor. The possibility that the structural conformation of the alpha 1-AT, rather than contamination with HSA, was responsible for its abnormal slower migratory rate was therefore tested. For this purpose alpha 1-AT preparations of different degrees of purification were submitted to heat treatment to induce a non-inhibitory conformation such as loop-sheet polymerization. Polymerization was followed both by the appearance in SDS- and non-denaturing PAGEs of high molecular weight bands, which were mostly present in less purified preparations of the inhibitor, and by a decrease in inhibitory activity. A higher degree of polymerization with complete loss of inhibitory activity was observed in the unprocessed alpha 1-AT preparation when dissolved in Na-phosphate buffer at acidic pH, and after dialysis. After heat treatment, the purified alpha 1-AT was shown to run faster in the gel and, in both reducing and non-reducing conditions, the calculated mass of the inhibitor was that expected of about 54 kDa. After reducing treatment, high molecular weight polymers in SDS-PAGEs were reduced, strongly suggesting that disulphide bridges are also involved in the polymerization of alpha 1-AT. Results indicate that the mobility of alpha 1-AT in SDS-PAGE is crucially dependent on its structural conformation which dictates the extent of SDS binding. HSA contaminating the alpha 1-AT preparation does not affect the inhibitor conformation although at a higher degree of contamination and in favourable conditions, it does reduce the inhibitor activity.