Franzen B, Carrubba C, Feingold D S, Ashcom J, Franzen J S
Biochem J. 1981 Dec 1;199(3):599-602. doi: 10.1042/bj1990599.
The catalytic-site thiol groups of UDP-glucose dehydrogenase from bovine liver were carboxymethylated with iodo[2-14C]acetate or with iodoacetamidofluorescein. After the residual thiol groups were carboxymethylated with iodoacetate, the proteins were digested with trypsin. The 14C-labelled peptide from the carboxymethylated enzyme was purified to homogeneity by successive thick-layer chromatography on silica gel, paper electrophoresis and chromatography, and column chromatography on Bio-Gel P-6. Homogeneous fluoresceincarboxamidomethylated peptide was prepared from a tryptic digest of fluoresceincarboxamidomethylated enzyme by specific adsorption--desorption from Sephadex G-25. The sequences of either peptide determined by the manual Edman dansyl procedure is: Ala-Ser-Val-Gly-Phe-Gly-Gly-Ser-Cys-Phe-Glx-Glx-Gly-Lys.
用碘代[2-¹⁴C]乙酸盐或碘乙酰胺荧光素对牛肝UDP-葡萄糖脱氢酶的催化位点巯基进行羧甲基化。在用碘乙酸对残留巯基进行羧甲基化后,用胰蛋白酶消化蛋白质。通过在硅胶上连续进行厚层色谱、纸电泳和色谱以及在Bio-Gel P-6上进行柱色谱,将羧甲基化酶的¹⁴C标记肽纯化至同质。通过从Sephadex G-25上进行特异性吸附-解吸,从荧光素羧甲基化酶的胰蛋白酶消化物中制备同质的荧光素羧酰胺甲基化肽。通过手动埃德曼丹磺酰法测定的任一肽的序列为:Ala-Ser-Val-Gly-Phe-Gly-Gly-Ser-Cys-Phe-Glx-Glx-Gly-Lys。