The subunit and polypeptide structure of hexosaminidases from human placenta

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Abstract
Previous reports have indicated that the hexosaminidases (HEX) are tetrameric enzymes composed of either two dimers of β chains (HEX B) or a β-chain dimer and an α-chain dimer (HEX A). HEX S contains only α chains. The apparent molecular weight of both α and β chains is 25 000. We isolated and purified HEX A and B more than 6000-fold. From HEX A we prepared HEX B and a more anodically migrating HEX S, which contained only α chains. After either extensive reduction and alkylation or performic acid oxidation, HEX B gave only a single protein band in polyacrylamide gel electrophoresis in sodium dodecyl sulfate; HEX A consistently gave two bands indicating it contained chains of two different molecular weights. Other reports note two bands in HEX A but have dismissed the high molecular weight band as a dimer caused by either hydrophobic interaction or an unbroken disulfide bond. To rule out hydrophobic interaction, we determined molecular weight by gel filtration in 6 M guanidine–HC1. To ensure complete disulfide bond breakage we used a harsher reduction and alkylation procedure than previously employed by others. The results were then confirmed by the use of two performic acid oxidation techniques, the stronger method resulting in extensive peptide bond oxidation. Samples of HEX A under reduction and alkylation or the weaker performic acid oxidation procedure, again chromatographed as two approximately equal peaks (50 000 and 25 000). HEX B, and the HEX B prepared from HEX A, resulted in one 25 000-dalton peak. HEX S chromatographed as a single 50 000 peak. We conclude that either the α chain in HEX has a molecular weight of 50 000 with one α chain per α subunit or that two 25 000-dalton chains are united by a nondisulfide crosslink (e.g., an isopeptide bond).