A combined nuclear magnetic resonance and absorbance stopped-flow apparatus for biochemical studies

A combined NMR and absorbance stopped-flow has been developed for monitoring the kinetics of biochemical reactions. We demonstrate its usefulness in following the alkaline denaturation of human hemoglobin. No glassblowing is required in the fabrication of the apparatus. Commercially available valves, syringes, tubing, and tubing connectors are employed whenever possible. Easily fabricated light guides are used to pipe light to and from the optical cell. The stopped-flow uses a 5-mm NMR tube followed by an optical cell with a 0.5-mm optical path length. This allows simultaneous measurements of NMR and absorbance changes. At a terminal flow velocity of 7.5 ml/s, the NMR and optical dead times were 60 and 260 ms, respectively. For the study reported here the oxyhemoglobin was labeled with a ¹⁹F probe attached to the β-93 cysteine. The native protein at pH 7 has an NMR spectrum consisting of a singlet, and the fully denatured hemoglobin sample at pH 12 has a spectrum consisting of three singlets. During the denaturation process another NMR peak appears rapidly and then decays away over the time course of the reaction. The absorbance changes at the high concentration employed for the NMR study (2.08 mm in heme) follow very nearly first-order kinetics. The events monitored by NMR, though in the same time frame as the optical changes, are of much greater complexity, and show the utility of multiple probes for monitoring protein unfolding.