Lozinsky Evgenia, Novoselsky Artem, Glaser Robert, Shames Alexander I, Likhtenshtein Gertz I, Meyerstein Dan
Department of Chemistry, Ben-Gurion University of the Negev, P.O. Box 653, Beersheba 84105, Israel.
Biochim Biophys Acta. 2002 Jul 3;1571(3):239-44. doi: 10.1016/s0304-4165(02)00257-x.
A fluorophore-nitroxide free radical dual-functional probe (FN) was utilized to study the kinetics of ascorbate (AH(-)) binding to Bovine Serum Albumin (BSA). Since the free radical fragment in the FN probe intramolecularly quenches fluorescence, ascorbate reduction of the nitroxide function is accompanied by a concomitant fluorescence intensity increase from the fluorophore. Thus, both fluorescence and the EPR techniques could be utilized to measure the reaction rate. In the presence of BSA protein, the observed rate of the overall process is the sum of that from at least two reactions: the reaction between free ascorbate and free probe, and the reaction between bound ascorbate and bound probe. Our findings show that the observed rate is strongly dependent on the ionic strength of the medium. A corollary of this observation is the indication of a purely electrostatic interaction between ascorbate and the BSA protein. This conclusion was further corroborated by 1H NMR measurement of the transverse relaxation time, T(2), of ascorbate protons in BSA solutions. Ascorbate ion was released from the ascorbate/BSA ensemble in the presence of increasing concentrations of NaCl. Binding constants of AH(-) to BSA were calculated at different ionic strengths at pH 7.4. Furthermore, an increase in ionic strength did not affect the ability of albumin to protect ascorbate against autoxidation. This suggests that the protein's protective antioxidant effect may be attributed to BSA binding of trace quantities of transition-metal cations (rather than ascorbate binding to BSA). This conclusion is supported by ascorbate UV-absorption measurements in the presence of albumin and Cu(2+) ions as a function of ionic strength.
一种荧光团-氮氧化物自由基双功能探针（FN）被用于研究抗坏血酸盐（AH(-)）与牛血清白蛋白（BSA）结合的动力学。由于FN探针中的自由基片段在分子内淬灭荧光，抗坏血酸盐对氮氧化物功能的还原伴随着荧光团荧光强度的相应增加。因此，荧光和电子顺磁共振技术都可用于测量反应速率。在BSA蛋白存在的情况下，观察到的整个过程的速率是至少两个反应速率之和：游离抗坏血酸盐与游离探针之间的反应，以及结合态抗坏血酸盐与结合态探针之间的反应。我们的研究结果表明，观察到的速率强烈依赖于介质的离子强度。这一观察结果的一个必然结果是表明抗坏血酸盐与BSA蛋白之间存在纯粹的静电相互作用。通过测量BSA溶液中抗坏血酸质子的横向弛豫时间T(2)，1H NMR进一步证实了这一结论。在NaCl浓度增加的情况下，抗坏血酸离子从抗坏血酸盐/BSA体系中释放出来。在pH 7.4的不同离子强度下计算了AH(-)与BSA的结合常数。此外，离子强度的增加并不影响白蛋白保护抗坏血酸盐免受自氧化的能力。这表明蛋白质的保护性抗氧化作用可能归因于BSA对痕量过渡金属阳离子的结合（而不是抗坏血酸盐与BSA的结合）。白蛋白和Cu(2+)离子存在下抗坏血酸的紫外吸收测量结果作为离子强度的函数支持了这一结论。
