Synthesis of a highly substituted N(6)-linked immobilized NAD(+) derivative using a rapid solid-phase modular approach: suitability for use with the kinetic locking-on tactic for bioaffinity purification of NAD(+)-dependent dehydrogenases.
This study is concerned with further development of the kinetic locking-on strategy for bioaffinity purification of NAD(+)-dependent dehydrogenases. Specifically, the synthesis of highly substituted N(6)-linked immobilized NAD(+) derivatives is described using a rapid solid-phase modular approach. Other modifications of the N(6)-linked immobilized NAD(+) derivative include substitution of the hydrophobic diaminohexane spacer arm with polar spacer arms (9 and 19.5 A) in an attempt to minimize nonbiospecific interactions. Analysis of the N(6)-linked NAD(+) derivatives confirm (i) retention of cofactor activity upon immobilization (up to 97%); (ii) high total substitution levels and high percentage accessibility levels when compared to S(6)-linked immobilized NAD(+) derivatives (also synthesized with polar spacer arms); (iii) short production times when compared to the preassembly approach to synthesis. Model locking-on bioaffinity chromatographic studies were carried out with bovine heart l-lactate dehydrogenase (l-LDH, EC 1.1.1.27), bakers yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) and Sporosarcinia sp. l-phenylalanine dehydrogenase (l-PheDH, EC 1.4.1.20), using oxalate, hydroxylamine, and d-phenylalanine, respectively, as locking-on ligands. Surprisingly, two of these test NAD(+)-dependent dehydrogenases (lactate and alcohol dehydrogenase) were found to have a greater affinity for the more lowly substituted S(6)-linked immobilized cofactor derivatives than for the new N(6)-linked derivatives. In contrast, the NAD(+)-dependent phenylalanine dehydrogenase showed no affinity for the S(6)-linked immobilized NAD(+) derivative, but was locked-on strongly to the N(6)-linked immobilized derivative. That this locking-on is biospecific is confirmed by the observation that the enzyme failed to lock-on to an analogous N(6)-linked immobilized NADP(+) derivative in the presence of d-phenylalanine. This differential locking-on of NAD(+)-dependent dehydrogenases to N(6)-linked and S(6)-linked immobilized NAD(+) derivatives cannot be explained in terms of final accessible substitutions levels, but suggests fundamental differences in affinity of the three test enzymes for NAD(+) immobilized via N(6)-linkage as compared to thiol-linkage.
Tynan
J
J
Department of Applied Biology and Chemistry, Institute of Technology Carlow, Kilkenny Road, Carlow, Ireland.
Forde
J
J
McMahon
M
M
Mulcahy
P
P
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification; D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification; D000818:Animals; D002417:Cattle; D016936:Gram-Positive Endospore-Forming Bacteria / Q000201:enzymology; D007770:L-Lactate Dehydrogenase / Q000138:chemical synthesis / Q000302:isolation & purification*; D042844:L-Lactate Dehydrogenase (Cytochrome); D009206:Myocardium / Q000201:enzymology; D009243:NAD / Q000031:analogs & derivatives / Q000737:chemistry* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D009243:NAD; D000426:Alcohol Dehydrogenase; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); D000594:Amino Acid Oxidoreductases; C073215:phenylalanine dehydrogenase (NAD)