Karakter Penghambatan SLPI Rekombinan Domain C terhadap Aktivitas Porcine Pancreatic Elastase

Abstract

Myocardial infarction is a chronic inflammatory condition characterized by myocardial cell necrosis due to reduced oxygen supply. In this condition, the increased activity of protease enzymes exacerbates tissue damage. Although the body naturally produces antiprotease enzymes such as Secretory Leukocyte Protease Inhibitor (SLPI), the endogenous levels are often insufficient in chronic inflammation. A biotechnology-based approach involving the production of recombinant SLPI, particularly its C-terminal domain (SLPIc). This study aimed to characterize the inhibitory kinetics of SLPIc against Porcine Pancreatic Elastase (PPE) using the specific substrate N-Succinyl-Ala-Ala-Ala-p-nitroanilide (SANA). SLPIc was produced in Escherichia coli BL21 (DE3) carrying the pGEX-4T-2- SLPIc plasmid, purified via Ni-NTA affinity chromatography, and quantified using the Bradford assay. Enzyme kinetics assays were conducted using Pocine Pancreatic Elastase (PPE) and varying concentrations of the N-Succinyl-Ala-Ala Ala-p-nitroanilide (SANA) substrate. The results demonstrated successful purification of SLPIc, evident by a single ~32-33 kDa protein band in the elution fractions with 307,5 until 370 mM imidazole. Kinetic analysis showed that SLPIc (66,5 μg/mL) decreased the Vmaks of PPE from 1,397 to 1,163 nmol/min and increased the Km from 1,501 to 2,322 mM, indicating a mixed-type inhibition mechanism. These findings highlight the therapeutic potential of SLPIc in mitigating tissue damage caused by excessive protease activity during myocardial infarction