Calf thymus histone as a substrate for neutral and acid proteases of leucocyte lysosomes and other proteolytic enzymes

Abstract

The use of calf thymus histone as a substrate has revealed a previously unknown neutral protease, optimally active at pH 7.2–7.3, in rabbit PMN lysosomes. The Sakaguchi reaction for the colorimetric determination of arginine has been modified, allowing a slow, linear development of color measurable at 500 nm over a period of up to 4 hr at 0–2°C. Specificity for arginine was shown since no other amino acid tested gave any color in the reaction. This new method has been used to measure arginine-reactive hydrolysis products released from histone by PMN lysosomes at neutral pH. Release of tyrosine, measured by the Folin method, was also used as an indicator of hydrolytic activity. Histone proved to be a useful substrate for the acid cathepsins of PMN, comparing favorably with hemoglobin, commonly used to measure such activity. Crystalline trypsin and chymotrypsin also hydrolyzed histone. The kinetics of arginine release by these enzymes over a period of 24 hr resembled those of neutral protease from PMN lysosomes.