Microsecond time-scale kinetics of transient biochemical reactions.

ABSTRACT: To afford mechanistic studies in enzyme kinetics and protein folding in the microsecond time domain we have developed a continuous-flow microsecond time-scale mixing instrument with an unprecedented dead-time of 3.8 ± 0.3 ?s. The instrument employs a micro-mixer with a mixing time of 2.7 ?s integrated with a 30 mm long flow-cell of 109 ?m optical path length constructed from two parallel sheets of silver foil; it produces ultraviolet-visible spectra that are linear in absorbance up to 3.5 with a spectral resolution of 0.4 nm. Each spectrum corresponds to a different reaction time determined by the distance from the mixer outlet, and by the fluid flow rate. The reaction progress is monitored in steps of 0.35 ?s for a total duration of ~600 ?s. As a proof of principle the instrument was used to study spontaneous protein refolding of pH-denatured cytochrome c. Three folding intermediates were determined: after a novel, extremely rapid initial phase with ? = 4.7 ?s, presumably reflecting histidine re-binding to the iron, refolding proceeds with time constants of 83 ?s and 345 ?s to a coordinatively saturated low-spin iron form in quasi steady state. The time-resolution specifications of our spectrometer for the first time open up the general possibility for comparison of real data and molecular dynamics calculations of biomacromolecules on overlapping time scales.

SUBMITTER: Mitic S 

PROVIDER: S-EPMC5626514 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature