Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria

New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of D-amino acids and immunochemical characterization of such conjugates.

Publisher Summary The chapter illustrates that the studies of hydrolytic agents have led to major advances in our knowledge of proteins, including the identification of the amino acid constituents of proteins and the development of the polypeptide concept of protein structure. These studies are now, for the most part, of historical interest, and provide an insight into protein hydrolysis. This is largely the result of three significant developments: (1) the discovery of chromatographic and electrophoretic methods for the examinatioin of the hydrolytic products of pure proteins; (2) the use of acids and enzymes as reagents for the systematic degradation of proteins into small peptides that are amenable to sequence analysis; and (3) the preparation of highly purified proteolytic enzymes that can be used for selective hydrolysis. The chapter mainly deals with the recent developments in the use of acids and enzymes for the hydrolysis of proteins, with particular emphasis on the hydrolytic methods, which are employed in amino acid sequence studies. Particular attention is given to those factors that must be considered in selection of a suitable hydrolytic reagent.

Hydrolysis of Proteins

The heat-induced aggregation of bovine β-lactoglobulin, dispersed in water at neutral pH and in different concentrations (10, 30, or 50 g of dry matter/L), was studied at 65 °C, and the results are related to a kinetic model. Native PAGE and SDS−PAGE analysis under nonreducing and reducing conditions showed that on heating disulfide-linked aggregates were formed and that the average size of these aggregates increased with increasing initial β-lactoglobulin concentration. In the presence of the thiol-blocking agent N-ethylmaleimide (NEM), at a molar ratio of NEM/β-lactoglobulin monomer of 1, all thiol groups were blocked and no disulfide-linked aggregates were formed, although with native PAGE high molecular mass noncovalently linked aggregates were observed. The formation of these aggregates accelerated with increasing NEM concentration until a molar ratio of NEM/β-lactoglobulin monomer of 1 was reached. In separate experiments we studied the effect of pH (in the range pH 6.0−8.0) on the aggregation of β-...

Heat-induced aggregation of beta-lactoglobulin: role of the free thiol group and and disulfide bonds

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Derek G. Smyth

Papers

Some Reactions of N-Ethylmaleimide1