Biological characterization of a Kunitz-type inhibitor from the Malaysian King cobra ( Ophiophagus hannah ) venom

Natural inhibitors are ubiquitous components, existing in a variety of organisms. Kunitz-type inhibitors (KTIs) are a group of proteins that bear remarkable homology to the Bovine Pancreatic Trypsin Inhibitor (BPTI) and have been extensively isolated and reported in snake venoms. The KTIs exhibit a wide variety of biological activities, including inhibition of various proteases, interference with hemostasis, inflammation, activation or blockade of acid-sensing ion channels (ASICs), and blockade of ion channels; showing their extensive functional diversity and biomedically relevant pharmacological properties. The king cobra (Ophiophagus hannah) is the longest venomous snake in the world, endemic from India through Southeast Asia. Members of the KTI family have been isolated and identify as either a weak chymotrypsin inhibitor or a trypsin/chymotrypsin inhibitor. This study aims to isolate and further characterize the pharmacological properties of a KTI from the Malaysian king cobra venom. The inhibitory effect on serine protease activity was determined using chromogenic substrates. The whole venom was partially purified employing size exclusion HPLC, and the isolated fractions were identified with N-terminal sequencing. The fractions (20 µg) were then incubated with plasmin (0.26 U) at 37 °C, at different times, and tested for inhibitory activities on its biological substrates. The trypsin activity towards its chromogenic substrate was 65% inhibited by the whole venom. After size exclusion chromatography, 13 fractions were isolated and were tested for inhibition of plasmin activity. The most remarkable effect was obtained with fraction 10, where a complete inhibition of the fibrinolytic activity of plasmin on fibrin plates was seen. Likewise, it partially inhibited the fibrino(geno)lytic activity of plasmin. These characterization studies will elucidate the biomedically relevant pharmacological properties intrinsic to a KTI from king cobra venom, which may lead to their potential use as therapeutic drugs and analytical tools for biomedical applications.