Biochemical characterization, stability studies and N-terminal sequence of a bi-functional inhibitor from Phaseolus aureus Roxb. (Mung bean).
Herein, we report the purification and biochemical characterization of a novel bi-functional protein proteinase/amylase inhibitor from the dietary leguminous pulse Phaseolus aureus Roxb. (Vigna radiata L.) by means of acetic acid precipitation, salt fractionation, ion-exchange chromatography (DEAE-cellulose) and affinity chromatography on trypsin-sepharose column. P. aureus inhibitor is a bi-functional inhibitor since it exhibits inhibitory activity towards trypsin-like and alpha-chymotrypsin-like serine proteinases as well as against alpha-amylases. It is a helix-rich protein (Mr 13,600) containing approximately eight tyrosines, one tryptophan and two cystines. N-terminal sequence alignment reveals no homology to other proteinase inhibitors reported from Phaseolus sp. thereby confirming that it is a novel inhibitor. Inhibitory activity measurements show that the inhibitor is quite stable even at extremely high temperatures and is only slightly affected by pH changes. Circular dichroism (CD) conformational studies revealed some changes in its near- as well as far-ultraviolet spectrum at extremes of pH and temperature. Treatments with trypsin for varying time periods did not alter its proteolytic inhibitory activity but caused some reduction in its amylase inhibitory activity.
Haq
Soghra Khatun
SK
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202 002, India.
Atif
Shaikh Muhammad
SM
Khan
Rizwan Hasan
RH
D000681:Amylases / Q000037:antagonists & inhibitors*; D002848:Chromatography, DEAE-Cellulose; D003553:Cystine / Q000032:analysis; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D027805:Phaseolus / Q000737:chemistry*; D011480:Protease Inhibitors / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000032:analysis; D014443:Tyrosine / Q000032:analysis
D004791:Enzyme Inhibitors; D011480:Protease Inhibitors; D014443:Tyrosine; D003553:Cystine; D014364:Tryptophan; D000681:Amylases