LDH-C4-substrate binary complexes studied by intrinsic fluorescence method.
Lactate dehydrogenase-C4 (LDH-C4) has been studied in presence of substrates using intrinsic fluorescence measurements. Excitation maximum of LDH-C4 occurred at 282 nm whereas fluorescence emission maximum was obtained at 340 nm. Fluorescence intensities at 340 nm showed that ligands viz. NAD+, NADH, pyruvate and lactate quench the relative fluorescence intensities of LDH-C4 in a concentration dependent manner. NAD+ and NADH produced a maximum quenching between 92-93% while pyruvate and lactate quenched the fluorescence of LDH up to 29% and 21% respectively. Association constants (Ka) based on fluorescence measurements were 6.05 x 10(4)M-1, 20 x 10(4)M-1, 0.113 x 10(4)M-1 and 0.3 x 10(4)M-1, for NAD+, NADH, lactate and pyruvate respectively. Stern-Volmer constants (Ksv) show that NAD+ and NADH have single Ksv of 4.07 x 10(4)M-1 and 1.47 x 10(5)M-1, whereas lactate and pyruvate indicated quenching reaction to be made up of two components. Ksv at low and high concentration of lactate respectively were 0.645 x 10(2)M-1 and 0.05 x 10(2)M-1, whereas corresponding Ksv with pyruvate were 1.008 x 10(3)M-1 and 0.408 x 10(3)M-1. Low Ksv at higher concentrations suggested that the aromatic chromophores are located within a hydrophobic environment. Red shift in fluorescence maximum (lambda max) by 2nm with lactate and 6nm with pyruvate showed that interaction of these ligands with LDH-C4 exposes some buried chromophores of the enzyme to the surface.
Gupta
G S
GS
Department of Biophysics, Panjab University, Chandigarh, India.
Kang
B P
BP
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D009243:NAD / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism
D007527:Isoenzymes; D009243:NAD; D014364:Tryptophan; D007770:L-Lactate Dehydrogenase