Activity Measurement and Multiplicity Detection of Human Secretory-Type Ribonuclease Based on Polycytidylic Acid/Ethidium Bromide Fluorescence

Abstract

Two analytical methods for human secretory-type ribonuclease, which are based on polycytidylic acid/ethidium bromide fluorescence, have been developed. The first is a method for measurement of secretory-type ribonuclease activity utilizing the radial diffusion of ribonuclease in a thin agarose gel plate containing polycytidylic acid and ethidium bromide. Ribonuclease activity was visualized as a dark circle on a fluorescent background under ultraviolet light after immersing the gel in a cooled acidic solution. The radius of the dark circle was proportional to the amount of the enzyme. This method allows quantitation of human secretory-type ribonuclease down to at least 5 × 10−5 unit, which corresponds to 60 pg. Secretory-type ribonuclease activity in 18 different human tissues and body fluids was measured. The second method is a zymogram technique for detection of secretory-type ribonuclease after isoelectric focusing, which includes placing a dried agarose film containing polycytidylic acid and ethidium bromide on the focused gel. Human secretory-type ribonuclease (less than 3 × 10−4 unit) was detected with a high band resolution on the same principle as that of the activity assay described above.