Stopped-flow front-face fluorometer: a prototype design to measure hemoglobin R----T transition kinetics.
Stopped-flow front-face fluorometer: a prototype design to measure hemoglobin R----T transition kinetics.
复制标题
停流正面荧光计：用于测量血红蛋白 R----T 转变动力学的原型设计。
DOI：
10.1016/0003-2697(89)90265-0
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发表时间：
1989
影响因子：
2.9
通讯作者：
Nagel,RL
中科院分区：
文献类型：
--
作者：
Hirsch,RE;Nagel,RL
停流技术已成功用于研究血红蛋白 (Hb) R → T 转变的动力学。我们之前使用正面荧光测定法证明 (i) Hb 的内在荧光主要源自 β37 Trp； (ii) 内在荧光对 R→T 转变敏感； (iii) 与 Hb 分子 (β93 Cys) 上特定位点结合的荧光探针的发射对 R → T 转变敏感。这些发现表明，停流前端荧光计可以探测特定位点的 R → T 转变，例如芳香族氨基酸和选择性结合外源荧光团的位点。我们开发了一种原型仪器，使用 Gibson-Durrum 停流装置作为核心，并使用改进的 Marquardt 算法进行数字数据分析系统。通过窄带通滤波器选择激发光（470 nm）和发射光（520 nm）。为了研究 R → T 转变，将与荧光探针 5-碘乙酰胺荧光素 (Hb A-AF) (1.0 g%) 共价结合的纯化氧 Hb A 溶液与含有 2 mg/ml 连二硫氨酸钠的脱氧缓冲液（pH 7.35，0.05 m 磷酸钾）快速混合。混合后最终浓度为 0.5 g% 的血红蛋白基本上完全是四聚体。观察到一级反应的速率常数接近 8 s−1，类似于氧 Hb A 报告的氧解离速率。总之，(i) 停流正面荧光计能够获得显着且可重复的数据，(ii) 外在荧光团 5-碘乙酰氨基荧光素与 Hb 的 β93 (Hb A-AF) 共价结合，可用作报告基团R → T 转变动力学。
Stopped-flow techniques are successfully used to study the kinetics of the R → T transition of hemoglobin (Hb). We have previously used front-face fluorometry to demonstrate that (i) the intrinsic fluorescence of Hb primarily originates from β37 Trp; (ii) the intrinsic fluorescence is sensitive to the R → T transition; and (iii) the emission of the fluorescent probes bound to specific sites on the Hb molecule (β93 Cys) is sensitive to the R → T transition. These findings suggested that a stopped-flow front-face fluorometer could probe R → T transitions at specific sites, such as the aromatic amino acids and sites selectively binding extrinsic fluorophores. We have developed a prototype instrument using as the core a Gibson-Durrum stopped-flow apparatus on line with a digital data analysis system using a modified Marquardt algorithm. Excitation (470 nm) and emission light (520 nm) were selected by narraw band pass filters. To study the R → T transition, a solution of purified oxy Hb A covalently bound to the fluorescent probe 5-iodoacetamidofluorescein (Hb A-AF) (1.0 g%) was mixed rapidly with deoxygenated buffer (pH 7.35, 0.05 m potassium phosphate) containing 2 mg/ml of sodium dithionine. The hemoglobin, at a final concentration of 0.5 g% after mixing, is essentially completely tetrameric. A first-order reaction was observed with a rate constant near 8 s−1, similar to the oxygen dissociation rate reported for oxy Hb A. In conclusion, (i) the stopped-flow front-face fluorometer is capable of obtaining significant and reproducible data, and (ii) the extrinsic fluorophore, 5-iodoacetamidofluorescein covalently bound to β93 of Hb (Hb A-AF), may be used as a reporter group of R → T transition kinetics.