John R. Cann

[12] Kinetically controlled mass transport of associating—dissociating macromolecules

Publisher Summary It has been emphasized that accurate interpretation of sedimentation patterns in terms of reaction mechanism requires the combined application of sedimentation with one or more other physicochemical methods. One such method is the relaxation technique for measuring the rates of rapid reactions and the chapter mentions the use of relaxation kinetics to distinguish among possible mechanisms for the dimerization of New England lobster hemocyanin induced by the binding of Ca 2+ . This chapter develops the subject of kinetically controlled association–dissociation reactions in a format that first considers theoretical predictions as to the shape of the sedimentation pattern and then turns to the combined application of velocity sedimentation, thermodynamic measurements, and relaxation kinetics to the characterization of the association–dissociation of hemocyanins from different species.

[10] Multibanded isoelectric focusing patterns produced by macromolecular interactions

Publisher Summary This chapter discusses multibanded isoelectric focusing patterns produced by macromolecular interactions. It highlights a phenomenological theory of isoelectric focusing of interacting systems that admits certain generalizations, two of which are particularly germaine to the routine analytical applications of isoelectric focusing. The first of these is that an amphoteric macromolecule, which complexes reversibly with several species of carrier ampholyte located at different positions along the isoelectric focusing column, can give a multimodal equilibrium pattern. This is based on the assumption that complex formation induces isomerization of the macromolecule with concomitant change in its isoelectric point (p I) . Each species of ampholyte induces a different isomer. To be as general as possible, the model presented in the chapter is silent as to whether or not the reversibly bound ampholyte might affect the p I of the isomer. It assumes that the several isomer–ampholyte complexes have electrophoretic mobilities and p I s, which differ from uncomplexed macromolecule and from each other. The calculations reveal that the number of peaks in the pattern depends upon the number of carrier ampholytes with which the macromolecule can form complexes, the strength of complex formation, and the concentrations of the several carrier ampholytes. The other generalization is that a macromolecule undergoing a sequence of q reversible pH-dependent conformational transitions can give an equilibrium isoelectric focusing pattern exhibiting q + 1 peaks, with the provison that the sequentially formed conformers have successively lower p I s.

[3] Effects of microheterogeneity on sedimentation patterns of interacting proteins and the sedimentation behavior of systems involving two ligands

Publisher Summary The chapter describes various model interactions that broaden the theoretical base for biophysical investigations into the architectural and regulatory roles played by protein association in vivo. A rather provocative result is that microheterogeneity with respect to association constant can give sedimentation patterns exhibiting three peaks, but other mechanisms can also give rise to trimodal patterns. These include protein associations mediated by two ligands, the system, monomer ⇄ trimer ⇄ nonamer, and slow reaction rates. In regard to conventional ultracentrifugal analysis, many of the patterns displayed herein bear a strong resemblance to patterns shown by mixtures of noninteracting proteins. Proof of inherent heterogeneity depends upon isolation of the various components. In a related vein, the results for self-association mediated by two ligands could conceivably have import for active enzyme sedimentation in solutions containing allosteric affectors, cofactors, or inhibitors, which promote association of the enzyme as in the case of carbamoylphosphate synthetase from E. coli .