A millisecond infrared stopped-flow apparatus.
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.
Tang
Jia
J
Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Gai
Feng
F
D003199:Computer Systems; D045304:Cytochromes c / Q000032:analysis* / Q000737:chemistry*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D017022:Flow Injection Analysis / Q000295:instrumentation* / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013055:Spectrophotometry, Infrared / Q000295:instrumentation* / Q000379:methods*
D045304:Cytochromes c