Purification of human plasma alpha 1-proteinase inhibitor and its inactivation by Pseudomonas aeruginosa elastase.
Human alpha 1-proteinase inhibitor was purified according to a modification of the method of Kurecki et al. (Anal. Biochem. 99, 415 (1979) ), with Affi-Gel Blue treatment before Zn-affinity column chromatography. The inhibitor was inactivated in the presence of Pseudomonas aeruginosa elastase (1/2,000 molar ratio) for 2 h at pH 7.5 and 25 degrees C. The inactivated inhibitor was purified by column chromatography on Sephadex G-75 and DE-52. Little or no difference was observed between the native and inactive inhibitors in immunological response, amino acid composition or far-ultraviolet CD spectrum. On the other hand, a considerable difference was observed in the near-ultraviolet CD spectrum. Two amino-terminal sequences were found in the inactive inhibitor in almost the same ratio; one was the same as that of the intact inhibitor and the other was Met-Ser-Ile-Pro-. The two components were separated by high-performance liquid chromatography using 0.1% trifluoroacetic acid containing 30-70% CH3CN (gradient) as the eluent. Amino acid analysis and N- and C-terminal amino acid sequence analyses indicated that one fraction corresponded to the sequence of 1-357 of the alpha 1-proteinase inhibitor and the other to 358-394. We concluded that P. aeruginosa elastase can inactivate human alpha 1-proteinase inhibitor by splitting the peptide bond of Pro357-Met358, leading to local change near the active site but little change in the structure as a whole. The split carboxy-terminal fragment binds tightly to the rest of the inhibitor.
D000596:Amino Acids / Q000302:isolation & purification; D001798:Blood Proteins / Q000302:isolation & purification* / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D010196:Pancreatic Elastase / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D000515:alpha 1-Antitrypsin
D000596:Amino Acids; D001798:Blood Proteins; D010446:Peptide Fragments; D000515:alpha 1-Antitrypsin; D010196:Pancreatic Elastase