Characterization of specific interactions of coenzymes, regulatory nucleotides and cibacron blue with nucleotide binding domains of enzymes by analytical affinity chromatography

Journal of Molecular Recognition : JMR
W C Thresher, H E Swaisgood

Abstract

The dissociation constant for the complex of rhodanese and Cibacron Blue, determined by analytical affinity chromatography using rhodanese immobilized on controlled-pore glass (CPG) beads (200 nm pore diameter) and aminohexyl-Cibacron Blue, was 44 microM which agreed well with the kinetic inhibition constant, suggesting that the dye binds at or near the active site of this enzyme. Formation of a binary complex of the dye and lactate dehydrogenase (LDH) was also characterized by direct chromatography of LDH on CPG/immobilized Cibacron Blue (KD = 0.29 microM). The binary complex formed between LDH and NADH was characterized by analytical affinity chromatography using both CPG/immobilized LDH and immobilized Cibacron Blue. Since the dye competes with NADH in binding to the active site of LDH, competitive elution chromatography using the immobilized dye allows determination of the dissociation constant of the soluble LDH.NADH complex. Agreement between the dissociation constants determined by direct chromatography of NADH on immobilized LDH (KD = 1.4 microM) and that determined for the soluble complex (KD = 2.4 microM) indicates that immobilization of LDH did not affect the interaction. Formation of various binary, ternary and quat...Continue Reading

References

Feb 1, 1976·Proceedings of the National Academy of Sciences of the United States of America·S T Thompson, E Stellwagen
Jan 30, 1979·Biochemical and Biophysical Research Communications·B H WeberD Piszkiewicz
May 11, 1978·Nature·J H PloegmanJ Russell
Jan 1, 1985·Methods in Enzymology·H F Fisher
Apr 1, 1973·The Biochemical Journal·R A Stinson, J J Holbrook
Dec 12, 1983·Biochimica Et Biophysica Acta·W C Thresher, H E Swaisgood

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Citations

Dec 1, 1992·Zentralblatt Für Bakteriologie : International Journal of Medical Microbiology·S E LindgrenW J Dobrogosz
Mar 2, 2011·Proteomics. Clinical Applications·Joao A PauloDarwin L Conwell
Jan 1, 2007·Proteomics. Clinical Applications·Rebekah L GundryRobert J Cotter
Jul 30, 2005·Proteomics·Nina ZolotarjovaWilliam C Barrett
Mar 1, 1992·Analytical Biochemistry·I ChaikenR Karlsson
May 5, 2007·Analytical Chemistry·Jeffrey C SmithDaniel Figeys

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