A Millisecond Infrared Stopped-Flow Apparatus

Authors: Tang, Jia; Gai, Feng

Source: Applied Spectroscopy, Volume 60, Issue 12, Pages 298A-334A and 1353-1494 (December 2006) , pp. 1477-1481(5)

Publisher: Society for Applied Spectroscopy

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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.

Keywords: CYTROCHROME C; INFRARED; KINETICS; PROTEIN FOLDING; STOPPED FLOW

Document Type: Research Article

DOI: http://dx.doi.org/10.1366/000370206779321328

Affiliations: Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Publication date: December 1, 2006

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