Abstract
An apparatus has been developed that allows photoaffinity ligands to be crossed-linked to milligram quantities of membrane proteins with maximum attainable yield following contact times of approximately 1 ms. The apparatus consisted of three parts: a conventional rapid mixing unit, a novel freeze–quench unit, and a photolabeling unit. The freeze–quench unit consisted of a rapidly rotating metal disk which was precooled in liquid nitrogen. Correct alignment of the exit jet from the sample mixer allowed up to 2 ml of sample to be frozen in a thin film on the disk. Experiments with colorimetric reactions showed the combined dead time of mixing and freeze–quenching to be submillisecond. Photoincorporation was maximized by prolonged irradiation of the freeze–quenched sample. Using this apparatus we determine the binding kinetics of the resting state channel inhibitor 3-[125I](trifluoromethyl)-3-(m-iodophenyl) diazirine (TID) to nicotinic acetylcholine receptor-rich membranes fromTorpedo.The binding kinetics for the125I-labeled α and δ subunits were biphasic; about half the binding was complete by 2.4 ms, and the remainder could be resolved and occurred with a pseudo-first-order rate constant determined at 4 μM [125I]TID of 12.0 ± 2.3 and 13.6 ± 4.0 s−1, respectively. This compares well to the same constant determined for the inhibition of agonist-induced cation flux inTorpedomembranes.
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