Interaction of pyridoxal-5'-phosphate and black-eye pea trypsin and chymotrypsin inhibitor.
Pyridoxal-5'-phosphate (pyridoxal-P) reacts with lysine residues of the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) at pH 7.4 and 30 degrees C, forming a Schiff-base which was stabilized by NaBH4 reduction. The interaction of pyridoxal-P and BTCI was accompanied by a difference spectrum presenting a negative peak at 382 nm and a positive peak at 435 nm which was shifted to 325 nm by reduction with NaBH4. The kinetics of interaction indicated that a noncovalent protein-pyridoxal-P complex was formed prior to Schiff-base formation. The number of pyridoxal-P (or pyridoxal) groups incorporated per protein molecule after NaBH4 reduction was determined spectrophotometrically as a function of the reagent/protein molar ratio. A saturation value of about 0.75 pyridoxal-P residues per protein molecule was found for an 80-100 molar excess of reagent over the protein. In the case of pyridoxal, the incorporation of reagent into the protein was much less than that of pyridoxal-P. The results obtained indicate that lysine residues in BTCI (5 per molecule) are in unfavourable environments for binding pyridoxal-P. The trypsin and chymotrypsin inhibitory activities of the pyridoxal-P-treated, NaBH4-reduced BTCI are slightly reduced.
D055598:Chemical Phenomena; D002621:Chemistry; D007700:Kinetics; D010940:Plant Proteins; D011732:Pyridoxal Phosphate; D012545:Schiff Bases; D013056:Spectrophotometry, Ultraviolet; D014361:Trypsin Inhibitors
D010940:Plant Proteins; D012545:Schiff Bases; D014361:Trypsin Inhibitors; C042736:trypsin, chymotrypsin inhibitor, black-eyed pea; D011732:Pyridoxal Phosphate