A dual wavelength scanning spectrophotometric method for the analysis of small absorbance changes in turbid suspensions is presented, which improves upon the point by point method of obtaining difference spectra in terms both of accuracy and rapidity. Examples are shown of the application of this instrument, in conjunction with a computer curve-resolving routine, to problems in photosynthetic electron transport.
The development of dual wavelength (double beam) spectrophotometry by Chance (1,2) made possible the measurement of absorbance changes smaller than 5 X 1O-4 units in turbid suspensions with high overall optical densities. This was accomplished by a differential technique in which the OD change at the wavelength of interest was compared with the OD change at a fixed reference wavelength. Typically, the wavelength of interest would be a reduced cytochrome a-band peak and the reference wavelength would be the closest possible isosbestic point between the reduced and oxidized forms of the cytochrome. Changes in sample turbidity that are relatively independent of wavelength, resulting from settling or from conformational changes of the particles, could thus largely be eliminated. Of lesser importance was the ability of the dual wavelength operating mode to compensate for fluctuations in emission from the light source.
Chance and Williams (3-5) used a dual wavelength spectrophotometer in their successful location, by steady state analysis, of the coupling sites in the mitochondrial cytochrome chain. Corresponding studies of cytochrome function in isolated chloroplasts have met with little success 1 Research carried out at Brookhaven National Laboratory under the auspices of the U. S. Atomic Energy Commission.