A Flow‐Quench Apparatus for Cryoenzymic Studies

Abstract

The flow-quench method was adapted to sub-zero conditions. Two apparatus were constructed: a rapid flow-quench apparatus (which take samples in the 5--300-ms time range) and a time delay flow-quench apparatus (0.5 s and longer). The apparatus were constructed so that the reagents are only in contact with chemically inert materials : glass, poly(tetrafluoroethylene) and poly(trifluorochloroethylene). The modified flow-quench apparatus were used to study the initial formation of creatine phosphate by creatine kinase at -15 degrees C in the time range 5 ms to 10 s. As at +4 degrees C [Travers, F., Barman, T. E. and Bertrand, R. (1979) Eur. J. Biochem. 100, 149--155], the time course of product formation was complex and consisted of three phase: a lag phase, a burst phase and the steady-state phase. The apparatus were also used to test chemically for reaction intermediates involving labile phosphate and phospho-enzyme complexes on the creatine kinase reaction pathway at -15 degrees C. Since neither type of intermediate could be detected down to 5 ms, this enzyme probably proceeds via a direct in-line type of mechanism.