Stopped-flow front-face fluorometer: a prototype design to measure hemoglobin R----T transition kinetics

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 beta 37 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 (beta 93 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 narrow 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 dithionite. 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.(ABSTRACT TRUNCATED AT 250 WORDS)