Determination of the operational molarity of solutions of bovine α-chymotrypsin, trypsin, thrombin and factor Xa by spectrofluorimetric titration

Abstract

Several esters of 4-methylumbelliferone and 2-naphthol were synthesized and examined as possible spectrofluorimetric titrants for bovine α-chymotrypsin, trypsin, thrombin, Factor Xa and for subtilisin Novo. 4-Methylumbelliferyl p-guanidinobenzoate hydrochloride (MUGB) is a satisfactory titrant for α- and β-trypsin, thrombin and Factor Xa and 4-methylumbelliferyl p-(NNN-trimethylammonium)cinnamate (MUTMAC) is a good titrant for α-chymotrypsin. The amount of enzyme used for spectrofluorimetric titration is 0.02–3.00nmol and the amount of 4-methylumbelliferone liberated is independent of the concentration of titrant and stoicheiometrically equal to the amount of enzyme used. Results obtained with MUGB and MUTMAC have been checked by spectrophotometric titration with p′-nitrophenyl p-guanidinobenzoate hydrochloride and p-nitrophenyl N2-acetyl-N1-benzylcarbazate respectively. p-Nitrophenyl N2-acetyl-N1-(9-anthrylmethyl)carbazate has been synthesized; it did not react with α-chymotrypsin. A satisfactory spectrofluorimetric titrant for subtilisin Novo was not discovered.