Triazine-dye affinity; chromatography.

functional resemblance of column groups to carbamoyl phosphate. The enzyme binds its two substrates, carbamoyl phosphate and L-aspartate, in an obligatory order, with carbamoyl phosphate leading (Grayson et al., 1979). If column groups are sufficiently similar to carbamoyl phosphate, the enzyme should be adsorbed more tightly in the presence of L-aspartate, owing to the ‘locking-on’ effect (O’Carra, 1978). Since no difference could be seen in the elution profiles containing Land D-aSpartate (Fig. 2), it is concluded that (a) the column groups do not resemble carbamoyl phosphate sufficiently to elicit the ordered-binding effect, and (6) column groups do not resemble L-aspartate. Both columns are therefore essentially non-biospecific in their adsorption of the enzyme. Carbamoyl phosphate has been shown to desorb the enzyme from a variety of mixed-function adsorbents, and evidence has been obtained to show that desorption is biospecific, i.e. involves the catalytically-significant enzyme-substrate complex (Yon, 198 I). Part of the evidence depends on comparing the desorbing capabilities of carbamoyl phosphate, P, and 2-glycerophosphate with their dissociation constants determined from steady-state kinetic and inhibition studies. These constants are 0.03 mM, I.OmM and 20mM respectively (Grayson et at., 1979). If desorption is biospecific, these compounds should be effective in the same order as their enzyme-ligand aftinities, i.e. carbamoyl phosphate > P, > 2-glycerophosphate. This expectation is confirmed for both adsorbents by the results shown in Fig. 3. However, the weaker ligands, P, and 2-glycerophosphate, were more effective in desorbing the enzyme from Blue Sepharose than from 10-CD-Sepharose; this was also true for carbamoyl phosphate at a much lower concentration (1 p ~ ) . Thus the adsorbent that binds the enzyme with lower affinity is also most responsive to biospecific desorption, in agreement with a theoretical prediction (Yon, 1980). It seems appropriate in the context of the present Colloquium to consider the relevance of this work to ‘mainstream’ affinity chromatography. Biospecific desorption is widely practised in ‘aftinity’ chromatography, and in the absence of firm evidence to the contrary, purifications are just as likely to be due to mechanisms (2)-(4) above as to mechanism (1). It is unfortunate that, after the initial (and still widely quoted) unambiguous definition of affinity chromatography by Cuatrecasas et al. (1968), the term has come to be used much more loosely; the literature contains many examples of ‘affinity chromatography’ in which the only certain biospecificity is in desorption, and biospecificity is adsorption is merely assumed. Certainly the time seems overdue for renewed consideration of the meaning we give to this term, and the title of the present Colloquium reflects, I hope, a willingness to come to grips with this problem.