A new method for the determination of α1-protease inhibitor ( α1-antitrypsin) phenotypes based on the formation of α1-protease inhibitor allele product·elastase complexes

Abstract

Up until now it has been assumed that the protease-binding property of α 1-protease inhibitor ( α 1PI) was destroyed by acid starch gel electrophoresis (pH 4.9). Analyses on acid starch gel blocks for pH and conductivity changes during and following a typical electrophoretic run showed that it was unlikely that the separating α 1PI would be exposed to pH values lower than 6.2, and that the allele products, following the passage of the buffer front, were in an environment of constant pH (6.3), extremely low conductivity and high field strength. These results strongly suggested the likelihood that α 1-PI would be chemically and physically unchanged as a result of exposure to acid starch gel electrophoresis. In order to test this likelihood, human serum was electrophoretically separated in acid starch gel and following electrophoresis, was immersed in 0.1 M diethylbarbiturate buffer, pH 8.6, containing 20 μ/ml of pancreatic elastase. The pH-adjusted (8.15) and elastase-impregnated starch gel layer was superimposed on hemoglobin-agar for 2.5 h at 37 °C followed by immersion of the hemoglobin-agar layer in 1% NaCl overnight, distilled water for 2 h, drying under filter paper and staining. The results showed zones of undigested hemoglobin indicating, unequivocally, that the separated α 1PI allele products are capable of forming complexes with proteases and that α 1PI is not inactivated following exposure to acid starch gel electrophoresis. Densitometric analysis of the transparent stained zones on a clear agar gel background offers an alternative to analysis of the acid starch gel-separated zones by antigen-antibody crossed electrophoresis and as such is suitable for identification of α 1-protease inhibitor phenotypes. Further, the method is specific for α 1PI and a densitometric scan provides direct information relative to the protease-binding capacity of the sample as well as the contribution of each α 1PI allele product to that capacity.