An Analysis of Affinity Chromatography Using Immobilised Alkyl Nucleotides

Abstract

Group specific matrices for affinity chromatography have now received considerable attention (1–9). Some of these studies have provided amongst the best examples of this aspect of affinity chromatography (10). Amongst the group specific matrices and in particular amongst the immobilised cofactors, the nicotinamide nucleotides present a very wide choice of applications and of different enzyme systems to study. However, because of the chemical complexity of the nicotinamide nucleotides, severe restrictions are imposed on the potential methods available for their immobilisation. These nucleotides may be immobilised readily onto cellulose or Sephadex or agarose containing aminocaproic acid (1,11,12). The resultant polymers are almost certainly of undefined nature (13) apart from being contaminated with unreacted carboxyl groups. The former need not necessarily be a drawback; the polymer preparation described by Allen & Majeries (10) does not specify which carboxyl groups of B_{1 2} are utilised in the linkage to the agarose and this undefined matrix is capable of purification factors of thousands. This is not so with nucleotides where the purification factors rarely exceed fifty times (1,8).