ABSTRACT
Investigations have been carried out on the complex formed between sorghum Inhibitor III and alpha-chymotrypsin by physico-chemical methods. An apparent dissociation constant (Ki) of 4.0 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 48,000 was estimated for the complex. The formation of the complex was accompanied by spectral changes in the 270--300 nm region of the spectrum. Preliminary evidence suggests that the sorghum Inhibitor III is structurally altered when it is incubated with alpha-chymotrypsin. Catalytically inactive derivative of alpha-chymotrypsin and trypsin, as well as their zymogens, did not interfere with the activity of the sorghum inhibitor towards the native enzymes. Sorghum Inhibitor I was shown to be a 'double-headed' inhibitor since it inhibits both trypsin and alpha-chymotrypsin independently.
Subject(s)
Edible Grain/analysis , Endopeptidases , Protease Inhibitors , Chromatography, Gel , Chymotrypsin/antagonists & inhibitors , Electrophoresis , Molecular Weight , Protease Inhibitors/isolation & purification , Spectrum Analysis , Trypsin , Trypsin InhibitorsABSTRACT
An investigation has been carried out on the proteinase inhibitors of grain sorghum (Sorghum bicolor (L.) Moench). One of the inhibitors has been isolated in a pure form and characterized. The proteinase inhibitor was extracted from the acetone-defatted sorghum meal and purified by selective thermal denaturation, ammonium sulfate fractionation, Sephadex gel filtration and DEAE-cellulose chromatography (DEAE-preparation II). This preparation was demonstrated to be a mixture of three inhibitor components by polyacrylamide disc gel electrophoresis. Further resolution of this mixture into Inhibitors I to III was achieved by QAE-Sephadex chromatography. Sorghum Inhibitor III was homogeneous by the criteria of disc gel electrophoresis and has been more fully characterized. A molecular weight of 25,000 was obtained for Inhibitor III by gel filtration and was in agreement with the value calculated from the amino acid composition of the inhibitor. The N-terminal amino acid residue of Inhibitor III, a single chain protein, was isoleucine. Sorghum proteinase inhibitors inhibit specifically the serine proteinases and are inactive towards the other classes of proteinases. Inhibitor III is primarily a chymotrypsin inhibitor, whereas Inhibitors I and II inhibit both trypsin and chymotrypsin.
