Microsecond time-scale kinetics of transient biochemical reactions

• DOI: 10.1371/journal.pone.0185888
• Publication Date: 2017-10-03T17:43:47Z
• Authors: Sandra Mitić; Marc J. F. Stramp...; Wilfred R. Hagen; Simon de Vries
• Keywords: Microsecond; Absorbance; Temporal resolution; Time constant

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