Quantitative affinity chromatography of adenosine deaminase on polymer-bound inosine: the assessment of binding constants by biospecific elution
Rosemeyer, H.; Seela, F.
Analytical Biochemistry 115(2): 339-346
ISSN/ISBN: 0003-2697 PMID: 7304964 DOI: 10.1016/0003-2697(81)90015-4
Affinity chromatography of adenosine deaminase (Ec 188.8.131.52.) on agarose-bound inosine with biospecific elution of the enzyme using linear gradients of adenosine or inosine leads via chromatographic parameters to a dissociation constant of the binary complex of Kdiss = 3.5 × 10−3m and to a binding enthalpy of ΔH = −3.9 kcal mol−1. These values can be explained by formation of two hydrogen bonds between immobilized inosine and the enzyme. The measurement of height equivalents of theoretical plates of the affinity column with dependence on the flow rate leads to the assumption that the velocity with which the equilibrium is reached is high compared with the flow rate; the high specificity of the affinity resin is not first of all due to a high number of theoretical plates but to the selectivity of the heterogenous enzymic reaction.