荧光猝灭法测定高粱原花青素-金属离子络合反应

原花青素是一种广泛存在于高等植物中具有生物活性的多酚类化合物,络合金属离子是原花青素的基本特性之一,不管是低聚体原花青素还是高聚体原花青素均能够络合金属离子。笔者研究利用金属离子与原花青素酚羟基的络合反应导致的原花青素(PC)荧光猝灭作用,研究高粱原花青素与金属离子的络合反应。移取2 900 μL醋酸盐缓冲溶液(pH 6)于1 cm石英池中,加入100 μL浓度为0.5 mmol/L高粱原花青素溶液,再逐次加入浓度为0.5 mmol/L金属离子(Al3+、Cu2+、Sn2+和Zn2+)溶液进行荧光滴定反应,每次加入金属离子溶液量为1 μL,金属离子加入总量为10 μL。记录原花青素的荧光强度衰变曲线,利用Stern-Volmer荧光猝灭方程计算得到4种金属离子Al3+、Cu2+、Sn2+、Zn2+分别与PC络合常数为1.35,0.32,1.44和0.29(×106 L/mol)。利用Job's法测定得到4种金属离子Al3+、Cu2+、Sn2+和Zn2+与PC形成的金属离子-PC络合物化学计量比(mol:mol)分别为1:2,1:2,1:1和1:2。此次对金属离子-PC络合反应定量关系的研究将有助于更好地了解原花青素的生理学功能。
Proanthocyanidins(epicatechin16(4？？8)catechin, PC)from Sorghum bicolor Moench are a class of polyphenol compounds widely distributed in higher plants with good physiological and pharmacological effects. It is well known that both procyanidin monomers and oligomers have the capacity to chelate with metal ions. However the studies on the metal-chelating capacity of proanthocyanidins with high molecular weight have been little reported hitherto due to the difficulty in obtaining these proanthocyanidins from the natural products. The fluorescence quenching effects of polyphenolic compounds epicatechin16(4？？8)catechin by metal cations Al3+, Cu2+, Sn2+ and Zn2+ were studied at pH 6 by measuring fluorescence quenching spectra. To measure fluorescence, 1 μL of 0.5 mmol/L metal ions solution were titrated into 100 μL of 0.5 mmol/L polyphenolic compounds solution in 2 900 μL pH 6 acetate buffer solution. The volume of metal ions solution was varied from 0 μL to 10 μL with the final concentration of 1.67 μmol/L. The results indicated that an obvious quenching effect on PC fluorescence intensity by Al3+, Cu2+, Sn2+and Zn2+ was observed, indicating the interaction between metal ions and PC. Continue adding more Sn2+, Al3+, Cu2+ and Zn2+ ions into solution caused a gradual decrease in the PC fluorescence intensity without changes of the maximum emission wavelength and shape of the peaks, which was interpreted that Al3+, Cu2+, Sn2+ and Zn2+ could quench the intrinsic fluorescence of PC. The Stern-Volmer equation was used to assessment of the Stern-Volmer constants(Ksv)between metal ions and PC. The results shown the metal Stern-Volmer constants of metal ions Al3+, Cu2+, Sn2+, Zn2+ for PC were 1.35, 0.32, 1.44, and 0.29(×106 L/mol), respectively. The stoichiometry of the metal ions binding to PC was determined using the method of continuous variations. Fluorescence quenching titration with metal ions have revealed that the stoichiometry of Al3+, Cu2+, Sn2+, Zn2+-bound PC were 1:2, 1:2, 1:1 and 1:2, respectively. These quantitative models of