Thompson S T, Stellwagen E
Proc Natl Acad Sci U S A. 1976 Feb;73(2):361-5. doi: 10.1073/pnas.73.2.361.
A simple, convenient, and sensitive spectrophotometric procedure is described for quantitative measurement of nucleoside phosphate binding sites constructed by the dinucleotide fold. The procedure involves difference spectral titration of such enzymes with the dye Cibacron blue F3GA in a spectral region remote from the intrinsic absorbance of proteins or natural ligands. The titration curves can be analyzed to determine the affinity of nucleoside phosphate binding sites for both the dye and the natural ligand over a potentially wide range of experimental conditions. The interaction of the dye with two proteins which contain the dinucleotide fold, lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) and phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3), is illustrated.
本文描述了一种简单、便捷且灵敏的分光光度法，用于定量测定由二核苷酸折叠构建的核苷磷酸结合位点。该方法涉及在远离蛋白质或天然配体固有吸光度的光谱区域，用染料汽巴克隆蓝F3GA对这类酶进行差示光谱滴定。通过分析滴定曲线，可在潜在的广泛实验条件下确定核苷磷酸结合位点对染料和天然配体的亲和力。文中举例说明了该染料与两种含有二核苷酸折叠的蛋白质，即乳酸脱氢酶（L-乳酸：NAD+氧化还原酶，EC 1.1.1.27）和磷酸甘油酸激酶（ATP：3-磷酸-D-甘油酸1-磷酸转移酶，EC 2.7.2.3）的相互作用。
