Sedimentation Behavior of Chymotrypsinogen A in the Vicinity of the Isoelectric Point

The techniques of velocity and equilibrium sedimentation have been used to determine conditions under which a well characterized protein, chymotrypsinogen A, already observed to exist as a monomer in certain media, will associate. Single, essentially symmetrical, schlieren patterns were observed in survey velocity experiments covering an extended concentration interval in the pH range 6.5 to 9.5, and at ionic strengths 0.002 to 3, in phosphate, Tris, ethylenediaminetetraacetate, and glycine buffers. The salts added to control the ionic strengths were LiCl, NaCl, KCl, and tetramethylammonium chloride. The temperature interval was 5–25°. Increases in the sedimentation coefficient were observed with decreasing ionic strength, suggesting association. The behavior of these coefficients was studied in detail at ionic strengths of 0.002, 0.03, and 0.1 at pH 9.30, which is in the vicinity of the isoelectric point. A value of 2.91 S was found for s020,w. Apparent molecular weights, calculated from equilibrium experiments under the same conditions, showed a marked concentration-dependent increase with decreasing ionic strength. This association is completely reversible, and, within the limits of experimental error, is unaffected by changes in temperature. It is suggested that electrostatic forces may play a significant role in the association at low ionic strength, although hydrogen and hydrophobic bonding cannot be precluded on the basis of present evidence.