A new method for the analysis of plasmin and plasminogen. Application of high-performance affinity chromatography.:application of high-performance affinity chromatography

概要

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Aminobenzamidine was immobilized via a spacer moiety to a micro-particle hydrophilic gel (Asahipak; Asahi Chemical Co.) and this affinity adsorbent was used for the separation of plasmin and plasminogen by high-performance affinity chromatography. The adsorbent was packed in a stainless steel column. When Glu-Plg was applied to the column equilibrated with phosphate buffer (pH 7.4), it was slightly retarded. Both Lys-Plg and Pl were retained on the column. Glu-Plg II was eluted more slowly than Glu-Plg I. The similar tendency was observed in Lys-Plg I and Lys-Plg II. Lys-Plg was eluted with ε-aminocaproic acid (EACA). For the elution of Pl, the coexistence of EACA and urea was necessary. This result indicates a two-site interaction of Pl with the immobilized ligand, i. e., at the lysine-binding sites and the catalytic site. Fluorometric detection of eluted protein and on-line assay of Pl activity using a fluorogenic substrate (Boc-Glu-Lys-Lys-MCA) revealed that effective separation of the enzyme could be achieved with high sensitivity (<10μg) within 1h. When the mixture of Glu-Plg, Lys-Plg and Pl was applied to the system, each of them could be eluted with the stepwise change of eluents. This system should be applicable in clinical diagnosis.
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