Chemical modification of the histidine residue located at the active site of ficin.
A fully active derivative of ficin was prepared by reversible blocking of its active thiol group with sodium tetrathionate and by oxidation of a methionine residue with sodium metaperiodate. Treatment of this active derivative with bromoacetone in the presence of 2 M urea, pH 6.5, led to its complete inactivation, and the sole change in its composition was the loss of one of its two histidine residues. A comparison of the peptide maps of pepsin digests of the bromoacetone-treated enzyme with its untreated control revealed the disappearance of one of the three histidine-containing peptides present in the control. This particular peptide was shown to have the sequence His-Ala-Val-Ala which is identical to the sequence around the histidine which can be cross-linked to the active thiol of ficin with dibromoacetone (Husain, S. S. and Lowe, G. (1970), Biochem. J. 117, 341). During the course of these studies a very sensitive assay for measuring the activity of ficin was developed which employs p-nitrophenyl-N-alpha-carbobenzoxy-L-citrullinate as the substrate.
Gleisner
J M
JM
Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul, 55100, USA.
Liener
I E
IE
D000085:Acetates / Q000737:chemistry; D000096:Acetone / Q000031:analogs & derivatives / Q000737:chemistry; D001671:Biochemistry / Q000379:methods*; D020134:Catalytic Domain; D002482:Cellulose / Q000737:chemistry; D002845:Chromatography / Q000379:methods; D005361:Ficain / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010100:Oxygen / Q000737:chemistry; D010206:Papain / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013455:Sulfur / Q000737:chemistry; D013885:Thiosulfates / Q000737:chemistry; D013997:Time Factors
D000085:Acetates; D010455:Peptides; D013438:Sulfhydryl Compounds; D013885:Thiosulfates; D000096:Acetone; C018235:bromoacetone; D006639:Histidine; D013455:Sulfur; C088674:dibromoacetic acid; D002482:Cellulose; D010206:Papain; D005361:Ficain; D010100:Oxygen