通过内在荧光法研究乳酸脱氢酶-C4-底物二元复合物。

LDH-C4-substrate binary complexes studied by intrinsic fluorescence method.

作者信息

Gupta G S, Kang B P

机构信息

Department of Biophysics, Panjab University, Chandigarh, India.

出版信息

Indian J Biochem Biophys. 1997 Jun;34(3):307-12.

PMID:9425751

Abstract

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.

摘要

已利用本征荧光测量法在底物存在的情况下对乳酸脱氢酶 - C4（LDH - C4）进行了研究。LDH - C4的最大激发波长出现在282nm处，而最大荧光发射波长为340nm。340nm处的荧光强度表明，配体即NAD⁺、NADH、丙酮酸和乳酸以浓度依赖的方式猝灭LDH - C4的相对荧光强度。NAD⁺和NADH产生的最大猝灭率在92 - 93%之间，而丙酮酸和乳酸对荧光的猝灭率分别高达29%和21%。基于荧光测量的缔合常数（Ka），NAD⁺、NADH、乳酸和丙酮酸分别为6.05×10⁴M⁻¹、20×10⁴M⁻¹、0.113×10⁴M⁻¹和0.3×10⁴M⁻¹。斯特恩 - 沃尔默常数（Ksv）表明，NAD⁺和NADH的单一Ksv分别为4.07×10⁴M⁻¹和1.47×10⁵M⁻¹，而乳酸和丙酮酸表明猝灭反应由两个成分组成。乳酸在低浓度和高浓度时的Ksv分别为0.645×10²M⁻¹和0.05×10²M⁻¹，而丙酮酸对应的Ksv分别为1.008×10³M⁻¹和0.408×10³M⁻¹。较高浓度下较低的Ksv表明芳香发色团位于疏水环境中。乳酸使荧光最大波长（λmax）红移2nm，丙酮酸使其红移6nm，这表明这些配体与LDH - C4的相互作用将酶的一些埋藏发色团暴露于表面。