Open AccessScreening and Purification of a Chymotrypsin Inhibitor from Entrolobium Saman Seeds
Author(s) -
Maher. A . Al.Maqtari,
Ahmed S. Saad
Publication year - 2010
Publication title -
maǧallaẗ ǧāmiʿaẗ al-sulṭān qābūs li-l-ʿulūm/sultan qaboos university journal for science
Language(s) - English
Resource type - Journals
eISSN - 2414-536X
pISSN - 2308-3921
DOI - 10.24200/squjs.vol15iss0pp19-29
Subject(s) - chymotrypsin , chemistry , sephadex , chromatography , trypsin , papain , size exclusion chromatography , kunitz sti protease inhibitor , isoelectric point , enzyme , biochemistry
A chymotrypsin inhibitor was isolated and purified from the seeds of Enterolobium saman (Leguminaceae family) by extraction with 100 mM phosphate buffer, heat treatment, ammonium sulphate precipitation, ion-exchange chromatography on DEAEcellulose and filtration through Sephadex G-75. The final preparation appeared to be homogeneous by both chromatographic and electrophoretic analyses. ESCI had a molecular weight of about 17,890 and an isoelectric point of 5.8. ESCI inhibited bovine chymotrypsin at an inhibitor-enzyme molar ratio of 1:2. The inhibition mode of chymotrypsin inhibitor was competitive on bovine chymotrypsin. Investigation has been carried out on the complex formed between chymotrypsin and chymotrypsin inhibitor by physico-chemical methods. An apparent dissociation constant (Ki) of 9.05 X 10-8 M has been calculated for the complex. This enzyme- inhibitor complex was isolated by gel filtration on Sephadex G-75 and a molecular weight of 43.000 was estimated for the complex. The inhibitor did not have any effect on other proteinases, such as papain, bromelin, elastase, α -amylase, trypsin and pepsin. The chemical modification of lysine residues indicated that –NH2 groups are not essential for the activity of ESCI toward chymotrypsin. The inhibitor was an acidic protein and was stable over a wide pH range of 2-12 and temperature range of 10o C-97o C.