Simultaneous measurements of fast optical and proton current kinetics in the bacteriorhodopsin photocycle using an enhanced spectrophotometer

Abstract

A one-of-a-kind high speed optical multichannel spectrometer was designed and built at NIH and described in this journal in 1997 [J.W. Cole, R.W. Hendler, P.D. Smith, H.A. Fredrickson, T.J. Pohida, W.S. Friauf. A high speed optical multichannel analyzer. J Biochem Biophys Methods 1997;35:16-174.]. The most unique aspect of this instrument was the ability to follow an entire time course from a single activation using a single sample. The instrument has been used to study rapid kinetic processes in the photon-driven bacteriorhodopsin photocycle and electron transport from cytochrome c to cytochrome aa3 and from cytochrome aa3 to oxygen. The present paper describes a second generation instrument with a number of important enhancements which significantly improve its capabilities for multichannel kinetic studies. An example application is presented in which the kinetics of photon-induced proton flow across the biological membrane is measured simultaneously with the individual steps of the photocycle determined optically. Matching the time constants for the two processes indicates which molecular transformations are associated with major proton movements.

Figure 1. Overall spectrometer system diagram

Figure 2. Diagram of a single analog channel

Figure 3. Data acquisition system (DAQ)

Figure 4. Block diagram of timing subsystem

Figure 5. Timing clocks

The duty cycles for the 100 kHz and 200 kHz sampling clocks were designed to meet the ADC manufacturer's specifications.

Figure 6. Trigger signals

The duty cycles of the Flashlamp and Q-switch signals were designed to meet the laser manufacturer's specifications. The time delay between the ADC Trigger and Q-switch is user selectable. All trigger signals are synchronous with the sampling clocks of Fig 5.

Figure 7. Fitting of proton current data

A. Raw data points using oriented gel (o); fit using five exponentials (line); raw data points using unoriented gel (x). B. Residuals of fit shown in panel A.