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将分子比为1:100的辣根过氧化酶(HRP)与N-(4-羧基环己烷甲基)马来酰亚胺的N-羟基琥珀酰亚胺酯(HECCM)反应,使HRP的氨基标上4-羧基环己烷甲基马来酰亚胺(Mal)基团而成HRP-Mal。反应在25℃pH7.0中搅拌60分钟,可使标记率达到每分子HRP标上一个Mal基团,HRP的回收率为83.3％。HRP-Mal再和等分子比的抗人肝癌铁蛋白Fab′在含有EDTA的pH6.0介质中4℃反应20小时,可使HRP和Fab′通过Mal交联生成HRP-Fab′交联物。后者通过凝胶过滤层析及SDS-聚丙烯酰胺电泳鉴定,分子量均为86,000,其中HRP和Fab′的分子比为1.03,均与理论值相符。交联物中HRP的活力可保留84.8％,酶蛋白和Fab′回收64％。
HRP reacted with N-hydroxysuccinimide ester (HECCM) of N- (4-carboxycyclohexanemethyl) maleimide at a molecular ratio of 1: 100, HRP Of the amino group is labeled with 4-carboxycyclohexylmethylmaleimide (Mal) group HRP-Mal. The reaction was stirred at pH 7.0 at 25 ° C for 60 minutes to achieve a labeling rate of one Mal group per molecule of HRP with an HRP recovery of 83.3%. Reaction of HRP-Mal with an anti-human hepatocellular ferritin Fab ’at an equimolar ratio for 20 h at 4 ° C in a EDTA-containing pH 6.0 medium allowed HRP and Fab’ to cross-link HRP-Fab ’cross-links via Mal. The latter was identified by gel filtration chromatography and SDS-polyacrylamide electrophoresis. The molecular weights of the latter were both 86,000. The molecular ratio of HRP to Fab ’was 1.03, both agree with the theoretical value. The viability of HRP in the cross-linked product can be kept at 84.8%, and the enzyme protein and Fab ’can be recovered at 64%.