Nadano D, Yasuda T, Sawazaki K, Takeshita H, Kishi K
Department of Legal Medicine, Fukui Medical School, Japan.
Anal Biochem. 1993 Jul;212(1):111-6. doi: 10.1006/abio.1993.1299.
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.
已开发出两种基于聚胞苷酸/溴化乙锭荧光的人分泌型核糖核酸酶分析方法。第一种是利用核糖核酸酶在含有聚胞苷酸和溴化乙锭的薄琼脂糖凝胶板中的径向扩散来测量分泌型核糖核酸酶活性的方法。将凝胶浸入冷却的酸性溶液后，在紫外光下，核糖核酸酶活性在荧光背景上呈现为一个暗圈。暗圈的半径与酶的量成正比。该方法可对人分泌型核糖核酸酶进行定量，最低可达至少5×10⁻⁵单位，相当于60皮克。测定了18种不同人体组织和体液中的分泌型核糖核酸酶活性。第二种方法是等电聚焦后检测分泌型核糖核酸酶的酶谱技术，该技术包括在聚焦凝胶上放置一张含有聚胞苷酸和溴化乙锭的干燥琼脂糖膜。按照与上述活性测定相同的原理，以高条带分辨率检测到人分泌型核糖核酸酶（小于3×10⁻⁴单位）。
