Equilibrium
dialysis (ED) has been employed by numerous investigators in the study of protein binding (l-5) and appears to be the most frequently employed technique. Shortcomings of this method have been reviewed by Goldstein (6) as well as Meyer and Guttman (7). Prolonged equilibration time and agit.ation may cause denaturation of the protein and foster bacterial growth. At low concentrations, as much as 20% of the ligand may be adsorbed to the dialysis bag and such adsorption may be irregular, varying from bag to bag (6).
A number of investigators (8-10) have employed eonal Sephadex gel chromatography in the st.udy of protein binding. According to Wood and Cooper (II), this technique is useful only in instances in which rate of dissociation of the protein-ligand complex is significantly slower than the elution rate. Since binding of ligands to proteins is usually rapidly reversible, this particular procedure appears to be limited in its application to the study of drug protein binding. The Hummel and Dryer (12) procedure, while circumventing this particular difficulty, requires careful select.ion of experimental condit'ions to re-establish the equilibrium baseline. In addition, integrat,ion of protein peak and ligand trough are laborious and imprecise, especially in those instances in which these areas are either diffuse or ill-defined.
Cooper and Wood (13) have described the use of Sephadex gel frontal analysis chromatography (GELFAC), which appears to overcome many of the shortcomings of previously described techniques. Although these investigators did provide data comparing binding results obtained via ED and GELFAC, t,he extent of the comparison was insufficient, to provide a definitive comparison of the two procedures.
In our own research, we have found that ED appears to be adequate 80 @ 1972 by Academic Press, Inc.
'Rcllco Co.. Vineland, N. J. ' Pharmacia (coarse grade)