Purification of Human Plasma α1-Proteinase Inhibitor and Its Inactivation by Pseudomonas aeruginosa Elastase

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Abstract
Human α 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°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-IIe-Pro-. The two components were separated by high-performance liquid chromatography using 0.1 % trifluoroacetic acid containing 30–70% CH 3 CN (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 α 1 -proteinase inhibitor and the other to 358–394. We concluded that P. aeruginosa elastase can inactivate human α 1 -proteinase inhibitor by splitting the peptide bond of Pro 257 -Met 358 , 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.

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