Temperature jump, laser instrument

Figure 5. Block diagram of a laser-heated temperature-jump instrument.

A near-infrared laser pulse at 1.54 pm was used to rapidly heat the aqueous peptide solution 10-20 °C, while a cw ultraviolet probe beam excited the fluorescence of the labeled peptide and monitored the relaxation kinetics. A schematic of the instrument is shown in Figure 1. To produce the temperature jump pulse the fundamental (1064 nm) of a Nd YAG laser (Continuum Surelite I), operating at 1.67 Hz, was focused with a 0.75 m lens into a one meter Raman cell (Princeton Optics, Inc.). The Raman cell contained 600 psi of CH4 and 500 psi of He and had a conversion efficiency of up to 20% for the first Stokes line (1.54 pm). The 1.54 pm wavelength... 

The laser temperature jump instrument can effectively be used to initiate and observe the fast events in protein/peptide folding and unfolding as well as those events that extend out to several milliseconds. In the present study, the unfolding of a helical peptide was determined to occur within tens of nanoseconds, supporting the need for nanosecond or faster initiation techniques. Promising results obtained by the laser temperature jump method will continue to stimulate the development of additional monitoring techniques such as UV absorption and circular dichroism. 

The LDH/NADH pyruvate ternary complex concentration is quite low, and it was found that the concentration of LDH/NADH + pyruvate equals approximately that of the LDH/NAD+ lactate. A temperature increase tips the equilibrium from right to left. Figure 15.10 shows the time-resolved fluorescence emission of NADH at 450 nm in response to a T-jump from 10 to 23 °C. There are two instrument response times one near 30 ns, which is the pulse width of the laser irradiation heating the sample, and the second is diffusion of heat out from the laser interaction volume that occurs around 15 ms (the latter response is not shown). Fitting the data (solid line) with a function of multiexponentials yielded four rates, as indicated on Fig. 15.10, in addition to these instrument response functions. 

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