Models for the analytical ultracentrifuge behavior of Helix pomatia alpha-hemocyanin. I. Experimental testing of...

The 'microheterogeneity model' (R.J. Siezen and R. van Driel, Biochim. Biophys. Acta 295 (1973) 131) and the 'incompetent whole molecule model' (G. Kegeles, Arch. Biochem. Biophys. 180 (1977) 530) for the dissociation of Helix pomatia alpha-hemocyanin whole molecules to half molecules were tested experimentally, using ultracentrifugation and stopped-flow dilution analysis. Results of differential sedimentation experiments followed by stopped flow analysis of separated fractions of 60 S and 100 S molecules were not entirely as predicted by the incompetent model, the agreement depending on the pH and ionic strength of analysis. A considerable amount of stopped-flow dilution response could be attributed to material sedimenting between 60 and 100 S. This material appears to be the main equilibrating fraction, and its amount is considerably larger than that predicted by the microheterogeneity model. Increased hydrostatic pressure was found to enhance this fraction, whereas fixation or low ionic strength reduced or eliminated this fraction. Nonequilibrium components of 30, 50 and 80 S were detected and partially purified by differential sedimentation.