{"title":"A Non-competitive Serpin-Like Thrombin Inhibitor Isolated from Moringa oleifera Exhibit a High Affinity for Thrombin","authors":"Sawetaji, Kamal Krishan Aggarwal","doi":"10.1007/s10930-023-10116-6","DOIUrl":null,"url":null,"abstract":"<div><p>The majority of the clotting factors involved in blood coagulation pathways are serine proteases and thrombin is one of the key serine proteases involved in blood clotting. Many synthetic and chemical drugs targeting these proteases as therapeutics are known. However, they are associated with serious side effects such as bleeding, haemorrhage, edema etc. Serine protease inhibitors from plants have been suggested as one of the potential anticoagulant molecules against thrombosis. In the present work, a direct thrombin inhibitor from <i>Moringa oleifera</i> was isolated, purified and characterized. The homogeneity of the inhibitor is confirmed on native- PAGE. The purified inhibitor (5 µg) showed 63% thrombin inhibition at pH 7.2 at 37 °C. The IC<sub>50</sub> value of the isolated inhibitor was determined as 4.23 µg. The inhibitor on SDS-PAGE appeared as a single protein-stained band corresponding to 50 kDa thereby indicating its molecular weight as 50 kDa. Purified thrombin inhibitor (5 µg) showed 12% inhibition of trypsin, and 17% inhibition of chymotrypsin. This suggests more specificity of purified inhibitor towards thrombin. The isolated inhibitor showed a non-competitive mode of inhibition against thrombin as determined by the Dixon plot. The inhibition constant (<i>Ki</i>) was calculated as 4.35 × 10<sup>–7</sup> M. The present work reports for the first time a direct thrombin inhibitor from <i>M. oleifera</i> which may be further explored as an antithrombotic drug.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":1.9000,"publicationDate":"2023-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"The Protein Journal","FirstCategoryId":"2","ListUrlMain":"https://link.springer.com/article/10.1007/s10930-023-10116-6","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
The majority of the clotting factors involved in blood coagulation pathways are serine proteases and thrombin is one of the key serine proteases involved in blood clotting. Many synthetic and chemical drugs targeting these proteases as therapeutics are known. However, they are associated with serious side effects such as bleeding, haemorrhage, edema etc. Serine protease inhibitors from plants have been suggested as one of the potential anticoagulant molecules against thrombosis. In the present work, a direct thrombin inhibitor from Moringa oleifera was isolated, purified and characterized. The homogeneity of the inhibitor is confirmed on native- PAGE. The purified inhibitor (5 µg) showed 63% thrombin inhibition at pH 7.2 at 37 °C. The IC50 value of the isolated inhibitor was determined as 4.23 µg. The inhibitor on SDS-PAGE appeared as a single protein-stained band corresponding to 50 kDa thereby indicating its molecular weight as 50 kDa. Purified thrombin inhibitor (5 µg) showed 12% inhibition of trypsin, and 17% inhibition of chymotrypsin. This suggests more specificity of purified inhibitor towards thrombin. The isolated inhibitor showed a non-competitive mode of inhibition against thrombin as determined by the Dixon plot. The inhibition constant (Ki) was calculated as 4.35 × 10–7 M. The present work reports for the first time a direct thrombin inhibitor from M. oleifera which may be further explored as an antithrombotic drug.
期刊介绍:
The Protein Journal (formerly the Journal of Protein Chemistry) publishes original research work on all aspects of proteins and peptides. These include studies concerned with covalent or three-dimensional structure determination (X-ray, NMR, cryoEM, EPR/ESR, optical methods, etc.), computational aspects of protein structure and function, protein folding and misfolding, assembly, genetics, evolution, proteomics, molecular biology, protein engineering, protein nanotechnology, protein purification and analysis and peptide synthesis, as well as the elucidation and interpretation of the molecular bases of biological activities of proteins and peptides. We accept original research papers, reviews, mini-reviews, hypotheses, opinion papers, and letters to the editor.