Microsecond time-scale kinetics of transient biochemical reactions

Microsecond time-scale kinetics of transient biochemical reactions

Mitic, S. (TU Delft BT/Biocatalysis)
Strampraad, M.J.F. (TU Delft BT/Biocatalysis)
Hagen, W.R. (TU Delft BT/Biocatalysis)
de Vries, S. (TU Delft BT/Biocatalysis)

2017

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 μs. The instrument employs a micro-mixer with a mixing time of 2.7 μs integrated with a 30 mm long flow-cell of 109 μm 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 μs for a total duration of ~600 μs. 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 τ = 4.7 μs, presumably reflecting histidine re-binding to the iron, refolding proceeds with time constants of 83 μs and 345 μs 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.

OA-Fund TU Delft

http://resolver.tudelft.nl/uuid:f736a8b4-3941-4de5-9116-126ddfe0538a

https://doi.org/10.1371/journal.pone.0185888

1932-6203

PLoS ONE, 12 (10)

Institutional Repository

journal article

© 2017 S. Mitic, M.J.F. Strampraad, W.R. Hagen, S. de Vries