A Millisecond Infrared Stopped-Flow Apparatus

Jia Tang, Feng Gai

Applied Spectroscopy

Abstract

In this paper, we have described a stopped-flow apparatus that is capable of measuring infrared kinetics in the amide I′ region of a protein’s vibrational spectrum. The dead time of this setup, determined by the reducing reaction of 2,6-Dichlorophenolindophenol by l-ascorbic acid, is between 6 to 15 ms, depending on the flow rate. Therefore, this stopped-flow IR method provides a means of measuring infrared kinetics in a time window that is not easily accessible to other mixing-based IR techniques. Using this apparatus, we have studied the alkaline transition of cytrochrome c and have found that this conformational event proceeds in a biphasic manner. The characteristic time constants of these two phases were determined to be 68 ± 20 ms and 624 ± 37 ms, respectively.

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