Evaluation of Gel Filtration Data on Systems Interacting Chemically and Physically L. W. KICHOL Russell Grimwade School of Biochemistry, University of Melbourne, Victoria, Australia A. G. OGSTOX Department of Physical Biochemistry, John Curtin School of Medical Research, Australian A’ational lin,iver,sity, Canberra, ;l.C.T., Australia AND D. J. WIKZOR C.S.I.R.O. Wheat Research Unit, North Ryde, New South Wales, Australia Received December 22, 1966; accepted January 6, 1967 A method is described for the evaluation of equilibrium constants from frontal gel filtration data on systems in which two dissimilar reactants interact physically as well as chemically to form a rapidly dissociable complex. Its application is demon- strated by an investigation on Sephadex G-100 of the interaction between ovalbumin and lysozyme at pH 5.8, ionic strength 0.02. Gel filtrat’ion of a syst,em in which two dis- similar molecules are in rapidly-established equilibrium with a single complex (A + B + C) yields an elut’ion profile which may be used bo determine the equilibrium constant, provided t,hat thermodynamic ideality in t,he solution phase is assumed. In earlier estima- tions of K (1, 2) it was assumed also that the partition coefficient of each species between the solution and the gel phases was inde- pendent of t,he composition of the former; this assumption is not necessarily justified, since a Johnst,on-Ogston effect (3) has been observed in the front)al gel filtration of several mixtures which do not, react chemi- cally in the solution phase (4). In a theoretical t’reat,ment of interactions in transport experiments (c>), a distinction was made between “chemical” and “physi- cal” interaction. In “chemical” interact’ions, t#he velocity of each chemical species was taken to be constant (independent of compo- sition), the constituent velocities depending only on the proportions of the various inter- acting species present. III “physical” inter- actions, the velocities of the individual species (whether these also interact chemi- cally or not) depend on composition, through the operation of, for example, frictional forces between their molecules. It has been shown (6) t,hat# the forms of elution boundaries in gel filtration can be expressed by equations entirely analogous t,o those for free transport (5). It was pointed out also that two types of interaction be- tween chemical species can occur: (i) t,here can be a dependence of the partition coefi- cient of each chemical species between free solution and gel on the composition of the free solution; and (ii) there can be chemical equilibria between species in t’he free solu- t.ion. Of these, (i) corresponds to “physical” and (ii) to LLchemical” interaction m the free-migrat,ion case, and the terms are used here in these senses. These correspondences 517