Proteolytic activity in bovine dental pulp

The autolysis of bovine dental pulp by endogenous proteolytic enzymes has been demonstrated by means of a colorimetric ninhydrin method. The activity is heat-labile and acts optimally at pH 3.0. Acid-denaturated hemoglobin proved to be a useful exogenous substrate for the cathepsins of bovine dental pulp. Optimal hemoglobin hydrolysis was shown to occur at pH 4.0. The activity was concentrated fivefold by heat fractionation under closely controlled conditions (pH 5.0, 56 ° C, 60 seconds). Further concentration of the activity was obtained by Sephadex G-75 filtration and hydroxyapatite column chromatography. The total hemoglobin-splitting activity present in bovine dental pulp was separated into four active fractions on the hydroxyapatite column. Two of the fractions differed considerably in their pH optima, which were observed to be at pH 3 and pH 5, respectively. The relative purification of the pH 3 enzyme was 80 when compared with the starting material.