LDH-C-4-SUBSTRATE BINARY COMPLEXES STUDIED BY INTRINSIC FLUORESCENCE METHOD

Lactate dehydrogenase-C-4 (LDH-C-4) has been studied in presence of su bstrates using intrinsic fluorescence measurements. Excitation maximum of LDH-C-4 occurred at 282mn whereas fluorescence emission maximum wa s obtained at 340nm Fluorescence intensities at 340nm showed that liga nds viz. NAD(+), NADH, pyruvate and lactate quench the relative fluore scence intensities of LDH-C-4 in a concentration dependent manner. NAD (+) and NADH produced a maximum quenching between 92-93% while pyruvat e and lactate quenched the fluorescence of LDH up to 29% and 21% respe ctively. Association constants (K-a) based on fluorescence measurement s were 6.05x10(4)M(-1), 20x10(4)M(-1), 0.113x10(4)M(-1) and 0.3x10(4)M (-1), for NAD(+), NADH, lactate and pyruvate respectively. Stern-Volme r constants (K-sv) show that NAD(+) and NADH have single K-sv of 4.07x 10(2)M(-1) and 1.47x10(5)M(-1), whereas lactate and pyruvate indicated quenching reaction to be made up of two components. K-sv at low and h igh concentration of lactate respectively were 0.645x10(2)M(-1) and 0. 05x10(2)M(-1), whereas corresponding K-sv with pyruvate were 1.008x10( 3)M(-1) and 0.408x10(3)M(-1). Low K-sv at higher concentrations sugges ted that the aromatic chromophores are located within a hydrophobic en vironment. Red shift in fluorescence maximum (lambda max) by 2nm with lactate and 6nm with pyruvate showed that interaction of these ligands with LDH-C-4 exposes some buried chromophores of the enzyme to the su rface.