The association of subunits of hog kidney D-amino acid oxidase apoenzyme was studied by analytical molecular sieve chromatography using the method of integral boundary experiments. The degree of subunit association was determined as a function of protein concentration over the range 3-900 μg/ml at eight temperatures between 4 and 20°. Over this range of conditions the apparent molecular weight as determined from column calibration increased with concentration and assumed values intermediate between monomer and dimer. The stoichiometry of association which provided a best fit to the experimental parameters was that of a dimerization reaction. The apparent dimerization constant was calculated at each temperature from the experimentally determined weightaverage partition coefficients measured at a series of protein concentrations. Investigation of the temperature dependence of subunit dimerization revealed a marked transition in the value of the association constant in the region of 12-14°, with the dimerization proceeding more favorably at temperatures above 14° and less favorably at temperatures below 12°. The molar enthalpy change for the association on either side of this transition was found to be approximately zero, but a value of 72 kcal/mole was found for ΔH over the transition range (12-14°). The pronounced variation of ΔH with temperature indicated a reversible change in heat capacity for the protein and strongly suggested that the protein might be undergoing isomerization reactions between different conformational states in addition to subunit association. Analysis of expected behavior for a system undergoing simultaneous dimerization and isomerization of subunits leads to the prediction that any measurement (e.g., optical spectra) capable of reflecting differences between different conformational isomers should exhibit concentration dependence at constant temperature. Evidence for the existence of such conformation isomers was found in the concentration dependence of the ultraviolet absorption, and optical rotatory dispersion, spectra of the protein. © 1969, American Chemical Society. All rights reserved.
