[8] Interfering and complicating adsorption effects in bioaffinity chromatography

The Synthesis of Several 8-Substituted Derivatives of Adenosine 5'-Monophosphate to Study the Effect of the Nature of the Spacer Arm in Affinity Chromatography

“…Yeast alcohol dehydrogenase displays little affinity for either adsorbent, supporting the contention that this dehydrogenase may prefer 6-linked rather than derivatives linked through other positions [I 7, 291. ~-Glyceraldehyde-3-phosphate dehydrogenase is known to demonstrate a considerable degree of nonbiological adsorption on binding to a NAD + derivative linked through a hydrocarbon spacer arm [9,30] but that this could be alleviated by introduction of a more hydrophilic spacer arm [17]. Fig.4 (F) confirms these observations ; the enzyme interacts very weakly with the more hydrophobic adsorbent and is completely unretarded by the hydrophilic adsorbent.…”

“…However, it is not clear whether these effects are related to the increased hydrophilicity of the arm, its increased rigidity endowed by the peptide linkage or to altered accessibility of the ligand to the macromolecule. Furthermore, these data [3,17] were obtained with adsorbents whose chemical synthesis was not rigorously defined. Unless, the coupling of a ligand to a preformed spacer arm/matrix assembly is essentially quantitative anh chemically unambiguous, the resulting adsorbent may still possess potentially reactive groups and the exact mode of binding of the ligand, and subsequently, the complementary enzyme, may be equivocal.…”

“…Two explanations for this behaviour are conceivable. Firstly, the interactions of the enzyme with the immobilised Ado-5'-P results from a synergistic addition of nonbiospecific interactions in- volving the spacer arm with weak bioaffinity [15,17]. Attempts have been made to assess the relative contributions of these effects [16] since such effects cannot be ruled out on theoretical grounds.…”

Purification of β‐galactosidase from wheat‐germ by affinity chromatography

“…For isolation of Escherichia coli fl-galactosidase Steers et al developed an adsorbent consisting of agarose gel coupled with /3-thiogalactoside as a iigand via spacer arms [i] . In the case of this adsorbant, however, the adsorption phenomenon was pointed out not to be ligand-specific and appropriate spacer arms devoid of the ligand could bind E. coli /I-galactosidase [3] . phenyl /3-D-glycoside, was used for isolation of glycosidases from bovine and murine liver, but resolution of the enzymes was difficult because these enzymes possessed similar affinity to the.binding glycones [4].…”

Biospecific interaction on solid phase between alcohol dehydrogenase and an immobilized NADH analogue studied by fluorometry

“…Nevertheless, little is known about the various parameters involved in the binding process per se on solid phase. For instance, does the enzyme interact with its immobilized complementary ligand by biospecific interaction or do non-specific factors such as ionic and hydrophobic interactions play a major role [2]? Different methods have been employed, especially in the area of general ligand affinity chromatography [3], to investigate whether and to what extent biospecific adsorption is involved in the affinitybinding step.…”