Open AccessA Flow‐Quench Apparatus for Cryoenzymic Studies
Author(s) -
BARMAN Thomas E.,
BRUN André,
TRAVERS Franck
Publication year - 1980
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1980.tb04880.x
Subject(s) - tetrafluoroethylene , creatine kinase , creatine , reagent , chemistry , phase (matter) , phosphate , materials science , analytical chemistry (journal) , chromatography , biochemistry , organic chemistry , copolymer , polymer
The flow‐quench method was adapted to sub‐zero conditions. Two apparatus were constructed: a rapid flow‐quench apparatus (which takes 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(trifluoro‐chloroethylene). The modified flow‐quench apparatus were used to study the initial formation of creatine phosphate by creatine kinase at –15°C in the time range 5 ms to 10 s. As at +4°C [Travers, F., Barman, T. E. and Bertrand, R. (1979) Eur. J. Biochem. 100 , 149–1551, the time course of product formation was complex and consisted of three phases: 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°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.