Design and geometry of the four-jet tangential ABAB micro-mixer.

<p>(a) Design of the micro-mixer body and the cuvette holder. (b) Geometry of the micro-mixer and the rectangular cuvette integrated in the plexiglass holder. Reaction components, A and B, enter the mixer body as indicated and subsequently flow through the four channels arranged in a cross. The components are then forced through a 100 μm wide and 50 μm deep μm Pt inlay mixing chamber (392.5 pL in volume and 3 mm in outer diameter). (c) Side-view slice through the mixer body and the Pt inlay along two opposite channels. (d) Top view of the channel arrangement and the premixing chamber of the dismounted mixer. The premixing chamber has dimensions of 100 x 100 x 50 μm³ yielding a volume of 500 pL.</p

Analysis of early stages in the re-folding of acid-denatured cytochrome <i>c</i>.

<p>Spectral components (a) and kinetic traces (b) of cytochrome <i>c</i> in a pH-jump (2 to 4.5) experiment obtained from singular value decomposition analysis. Time zero (t₀) at pH 2 (blue), I₁ (magenta), I₂ (red), I₃ (green). The spectrum of native protein (nat) at pH 6 (black) was obtained after prolonged incubation. (c) Interpretational scheme of the experiment as described in the text.</p

Microsecond time-scale kinetics of transient biochemical reactions

<div><p>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 <i>c</i>. 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.</p></div