Volume Changes in the Isoelectric Heat Aggregation of Serum Albumin

Abstract

High precision dilatometry and buoyancy measurements in a quartz spring balance show that the change of partial specific volume of serum albumin accompanying aggregation a t the isoelectric point is positive, and of the order of A V = + i . 2 -+ 0.1 pl/g protein. Experiments a t high hydrostatic pressure ( < 3000 atmospheres) confirm this result, the aggregation reaction being suppressed significantly.As it appears by measurements of optical rotatory dispersion that the molecular secondary structure is kept essentially unchanged during the reaction, it is suggested that the water of hydration of the monomers, which probably is in a state of high density due to electrostriction, is partially eliminated from the molecular surface in the state of aggregation.The action of heat on globular proteins leads to conformational changes (denaturation) and/or changes of particle size (aggregation). As shown by kinetic experiments, the correlation between t,he two processes depends on the inter-and intramolecular electrostatic potential of the charges on the polyampholytic molecules [1,2], maximum rate of aggregation being observed in the isoelectric point, while maximum denaturation occurs a t high positive or negative net charge of the molecules. As a consequence one might expect the "isoelectric coagulation" [3,4] to represent a polymerization reaction based on unaltered native molecules. I n fact, spectroscopic results [2] substantiate this concept, suggesting a phase separation rather than conformational changes of the reacting monomers to be involved in the coagulation process.I n order to characterize the state of the isolated (monomeric) molecule as compared to the coagulated system, differences in conformation and in the solvent-solute interactions have to be analyzed. I n this context, volume change data may provide useful criteria for structural alterations in both the solvent and the solute or for changes in the solvent-solute interactions [5].The following results are based on dilatometry and water release experiments in a quartz spring balance. I n addition experiments at high hydrostatic pressure are presented. Parallel conformational analyses are performed using optical rotatory dispersion spectroscopy. MATERIALS AND METHODSBovine serum albumin of highest purity (Sigma, crystallized and lyophilized, and Behring-Werke, Dedicated to Professor F. Lynen on occasion of his sixtieth birthday. trocken, reinst) was used without further purification. Tobacco mosaic virus protein from the common strain of tobacco mosaic virus was prepared according to [6]. Reagents were of A-grade purity; bidistilled water was used throughout. Buffers were deaerated by heating to 90 "C or (in case of protein solutions) by keeping the solutions under reduced pressure for several minutes. Protein concentration (cp) was calculated from the absorbance a t 280 nm and from dry weight determination after exhaustive dialysis against water.The degree of aggregation was estimated from light-scattering [4], centrifugation and ultracentrifugation ...

Reconstitution of lactic dehydrogenase from pig heart after reversible high-pressure dissociation

“…In this connection, the previously mentioned exposure of interior hydrophobic residues or domains of the protein to the polar solvent may be important. Since hydrophobic interactions are weakened by high pressure (Kauzmann, 1959), aggregation during pressure incubation is inhibited (Jaenicke, 1971).…”

Application of High Hydrostatic Pressure to Dissociate Aggregates and Refold Proteins

“…In a specific example, thermally-induced aggregates of human serum albumin resulted in an increase in the protein specific volume of (78 +/-7 ml/mole) [22]. Protein aggregates are frequently less dense than native proteins.…”