Studies On Purification And Quantitation Of Carboxypeptidase Inhibitor From Potato Tubers

Potato tuber contains a kind of proteinase inhibitor, which is namedcarboxypeptidase inhibitor (CPI) with a molecular weight of approximately 4200Da.CPI is a specific inhibitor of the carboxypeptidase B (CPB) from human plasma invivo. CPB, also named as thrombin-activatable fibrinolysis inhibitor (TAFI), could beactivated and converted to TAFIa that produce downregulatively plasmin. TAFIa isable to interfere the initiation of fibrinolytic system. Because of CPI is an inhibitor ofTAFIa, CPI would become a potential drug for assistant thrombolytics which isprevent to the limited safeten dose of traditional thrombolytics.A rapid, stable method for estimating the biological activity of CPI wasdeveloped. The concentration of Carboxypeptidase A (CPA) and substrate were 16mg/L and 1mmol/L on the method, respectively. The sensitivity was 1.04mg/L, andthe linear response ranged from 1.85 ⁶.63mg/L on the method.A radioimmunoassay of CPI (CPI-RIA) was developed with the secondantibody-PEG method. The antiserum against at CPI was obtained by immunizing therabbits with CPI, and ¹²⁵I-CPI was prepared by the chloramines-T method. The intra-and inter-assay CV of CPI-RIA was 10.2% and 7.4%, respectively. The sensitivitywas 1.5μg/L and the average recovery was 104%. The antiserum of CPI provided alinear response from 7 ¹04μg/L, and ED₅₀ on the assay was 17.1μg/L.An isolated and purified method of CPI from Potato was established. Theprocess such as extracting media, concentration, ion-exchange and gel filtrationchromatography was optimized with analyzed the biological activity or CPI-RIA.About 20 mg CPI was obtained from 320g fresh potato, and the purity of CPI wasover 90%. The recovery rate of CPI from potato on this isolated method was 50.9%.The method is able to isolate or purify CPI by rapid and commercial technology, andit is useful to isolate CPI with large-scale.