Activity measurement and multiplicity detection of human secretory-type ribonuclease based on polycytidylic acid/ethidium bromide fluorescence.
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 x 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 x 10(-4) unit) was detected with a high band resolution on the same principle as that of the activity assay described above.
Nadano
D
D
Department of Legal Medicine, Fukui Medical School, Japan.
Yasuda
T
T
Sawazaki
K
K
Takeshita
H
H
Kishi
K
K
D001826:Body Fluids / Q000201:enzymology; D004058:Diffusion; D004591:Electrophoresis, Polyacrylamide Gel; D004996:Ethidium; D005069:Evaluation Studies as Topic; D005260:Female; D006801:Humans; D007525:Isoelectric Focusing; D008297:Male; D011066:Poly C; D011247:Pregnancy; D012260:Ribonucleases / Q000032:analysis* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D014018:Tissue Distribution
D011066:Poly C; D012260:Ribonucleases; D004996:Ethidium