A versatile low-temperature stopped-flow instrument compatible with both rapid and slow scanning spectrometers
✍ Scribed by Harold E. Van Wart; John Zimmer
- Publisher
- Elsevier Science
- Year
- 1981
- Tongue
- English
- Weight
- 768 KB
- Volume
- 117
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A low-temperature stopped-flow instrument has been constructed which consists of a Teflon stopped-flow module and a quartz observation cell that are held between two halves of a contoured brass cooling block. The temperature of the system is controlled by pumping precooled methanol through channels in the cooling block, and the exposed surfaces of the observation cell are kept free of frost with a stream of precooled dry nitrogen. The instrument is versatile, compact, and can be operated over the range -55 to 25°C without leaks or optical artifacts in three modes. First, it serves as a conventional stopped-flow instrument using light of fixed wavelength to follow the progress of the reaction. In the second mode, the instrument is interfaced to a rapid scan spectrometer that uses an intensified photodiode array detector and optical multichannel analyzer to obtain time-resolved spectra of reaction intermediates. The absorption spectra of compounds I and II formed during the reaction of horseradish peroxidase with hydrogen peroxide are time-resolved at 20.2'C in methanol/phosphate buffer (60/40, v/v%). In the third mode, the peroxidase reaction is studied at -51.4"C and the absorption spectra of the intermediates are obtained with a Varian Model 219 spectrophotometer. Since the stopped-flow instrument is compact, it is also compatible with the sample compartments of other slow-scanning laboratory spectrometers and it should facilitate the study of reaction intermediates by other forms of optical spectroscopy.