D'Azzo A, Hoogeveen A, De Wit-Verbeek H A
Clin Chim Acta. 1978 Aug 15;88(1):1-7. doi: 10.1016/0009-8981(78)90140-7.
A simple and rapid microfractionation procedure is described which enables the separate analysis of hexosaminidase A and B activities in as little as a few hundred to a thousand cultured human cells. 25 microliter cell homogenate is added to a pellet of DEAE-cellulose and 50 microliter 0.1 M NaCl in buffer. After centrifugation the hex B and I forms are measured in the supernatant, whereas hex A is determined by direct incubation of the DEAE pellet with methylumbelliferyl substrate. The reliability and reproducibility of the method is compared with that of heat inactivation and column chromatography. The application of the procedure is illustrated by analyses of fibroblasts and cultured amniotic fluid cells from pregnancies at risk for Tay-Sachs disease and by serum assays for the diagnosis and heterozygote testing of this disease.
本文描述了一种简单快速的微量分级分离程序，该程序能够在仅几百至一千个培养的人类细胞中分别分析己糖胺酶A和B的活性。将25微升细胞匀浆加入到DEAE-纤维素沉淀和50微升缓冲液中的0.1 M NaCl中。离心后，在上清液中测量己糖胺酶B和I型，而己糖胺酶A则通过将DEAE沉淀与甲基伞形酮底物直接孵育来测定。将该方法的可靠性和可重复性与热灭活和柱色谱法进行了比较。通过对患泰-萨克斯病风险妊娠的成纤维细胞和培养羊水细胞的分析以及对该疾病的诊断和杂合子检测的血清检测，说明了该程序的应用。
