Purification and characterization of serine proteinase inhibitors from two South African indigenous plants, Acacia karoo and Acacia schweinfurthii

Abstract

Serine proteases are known to perform a wide range of functions essential to life; however there has to be some form of control mechanism in place. One of the many control mechanisms is their specific inhibition by protein proteinase inhibitors. Proteinase inhibitors in plants, present in their seeds, participate in defense mechanisms and their production is induced by herbivory or wounding. Plant proteinase inhibitors have been reported to inhibit a variety of serine proteinases, including enzymes of the blood coagulation cascade. In this study, various indigenous seed extracts were screened for potential serine proteinase inhibition. Acacia schweinfurthii was selected as a potential inhibitor that inhibited trypsin and factor X. The AS inhibitor was successfully purified to homogeneity by precipitating with 80 percent (v/v) acetone and the sequential chromatographic steps including ion-exchange chromatography, size exclusion chromatography, affinity purification on a trypsin-agarose column and RP-HPLC. Reducing SDS-PAGE conditions revealed an inhibitor of two polypeptide chains A and B of approximate molecular weights 16 and 10 kDa, respectively, and under non-reducing conditions, 25 kDa was observed. The inhibitor was shown to inhibit trypsin, chymotrypsin and factor X indicating the dynamic nature of the reactive site. An enzyme: inhibitor ratio of 1:1, and a Ki of 3.45nM was determined for the AS inhibitor on trypsin, and the inhibitor also weakly inhibit chymotrypsin. AS inhibitor and STI inhibited factor X with a Ki values of 13.7nM and 77.5μM respectively. Amino acid analysis revealed Mmin values of the A- and B- chain of 15,000 and 7,800, respectively. The effect of seed extracts on the activated partial thrombin time (APTT) and prothrombin time (PT) was tested. No prolongation of the PT was obtained. For the crude extracts of AK and AS, IC200 values of 4.6 and 189.62 μg/mL, were respectively obtained. For the purified fractions of STI, AS and AK, IC200 values of 51.5, 114.31 and 893.8 μg/ml were observed, respectively. Keywords: proteinase inhibitors, Acacia species, trypsin inhibitor, FX inhibitor