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Intermolecular Cross-linking of Monomeric Proteins and Cross-linking of Oligomeric Proteins as a Probe of Quaternary Structure

1972; Elsevier BV; Volume: 247; Issue: 17 Linguagem: Inglês

10.1016/s0021-9258(20)81144-2

1083-351X

Frederick H. Carpenter, Kathryn Tinker Harrington,

Enzyme Structure and Function

Abstract Reaction of leucine aminopeptidase with dimethyl suberimidate at pH 8.5 and room temperature, followed by incubation with sodium dodecyl sulfate and mercaptoethanol and then electrophoresis in 3.5% acrylamide gels resulted in six protein bands with molecular weights as integer values of 56,000. Under reaction conditions where appreciable cross-linking took place, the species corresponding to dimers, tetramers, and hexamers were much more prevalent than those corresponding to trimers and pentamers, the latter being barely detectable. Providing that the ability of the protomers to form cross-links reflects their spatial arrangement in the oligomer, the results suggest that leucine aminopeptidase is composed of six identical protomers arranged as a trimer of dimers. Of the possible arrangements for a hexamer, the planar hexamer with all heterologous interactions is incompatible with these data. Thus, cross-linking reactions may yield information on the spatial arrangement of protomers in oligomers as well as on the number and kinds of protomers in the oligomer. Individual treatment of a number of monomeric proteins with dimethyl suberimidate in the frozen state at -10° in the presence of sodium dodecyl sulfate and mercaptoethanol yielded intermolecular cross-linked species which could be separated into a large number of components on gel electrophoresis in sodium dodecyl sulfate. For any one cross-linked protein the plot of logarithm of molecular weight against Rf fell on a straight line for Rf values between 0.2 and 0.8. The lines described by different proteins, although generally having similar slopes, were not always superimposable. This procedure for cross-linking monomeric proteins has potential use in determining molecular weights by gel electrophoresis in sodium dodecyl sulfate.