Sogawa K, Takahashi K
J Biochem. 1978 Jun;83(6):1783-7. doi: 10.1093/oxfordjournals.jbchem.a132094.
Conditions have been investigated for the use of fluorescamine-labeled casein as a substrate for fluorometric assay of proteinases. Fluorescamine-labeled casein can be prepared simply by mixing solutions of casein and fluorescamine at pH 8.0 and used without removal of the excess reagent or its hydrolysis product. The fluorescence of the labeled casein and its enzymatic digest is moderately stable in the range of pH 7.0 to 10.0. Activities can be determined by measuring the fluorescence of the hydrolysis products soluble in 0.1 M trichloro acetic acid solution at pH 4.0 after adjusting the pH of the acid-soluble fraction to 7.7. This method is suited for assay of proteinases active at neutral to slightly alkaline pH values, and is capable of quantitating about 0.05 microgram of trypsin or 0.5 microgram of alpha-chymotrypsin or papain. The assay can be done in the presence of large amounts of contaminating amino acid, protein and/or exopeptidases which may interfere with the ordinary assay of proteinases.
已对使用荧光胺标记的酪蛋白作为蛋白酶荧光测定底物的条件进行了研究。荧光胺标记的酪蛋白可通过在pH 8.0下将酪蛋白溶液和荧光胺溶液简单混合来制备，且无需去除过量试剂或其水解产物即可使用。标记酪蛋白及其酶解产物的荧光在pH 7.0至10.0范围内适度稳定。在将酸溶性部分的pH值调至7.7后，通过测量可溶于pH 4.0的0.1 M三氯乙酸溶液中的水解产物的荧光来测定活性。该方法适用于测定在中性至微碱性pH值下具有活性的蛋白酶，能够定量约0.05微克的胰蛋白酶或0.5微克的α-糜蛋白酶或木瓜蛋白酶。该测定可在存在大量可能干扰蛋白酶常规测定的污染氨基酸、蛋白质和/或外肽酶的情况下进行。
