In a previous paper (Ramsay and Eftink, Biophys. J. 66:516-523) we reported the development of a modified spectrophotometer that can make nearly simultaneous circular dichroism (CD) and fluorescence measurements. This arrangement allows multiple data sets to be collected during a single experiment, resulting in a saving of time and material, and improved correlation between the different types of measurements. The usefulness of the instrument was shown by thermal melting experiments on several different protein systems. This CD/fluorometer spectrophotometer has been further modified by interfacing with a syringe pump and a pH meter. This arrangement allows ligand, pH, and chemical denaturation titration experiments to be performed while monitoring changes in the sample's CD, absorbance, fluorescence, and light scattering properties. Our data acquisition program also has an ability to check whether the signals have approached equilibrium before the data is recorded. For performing pH titrations we have developed a procedure which uses the signal from a pH meter in a feedback circuit in order to collect data at evenly spaced pH intervals. We demonstrate the use of this instrument with studies of the unfolding of sperm whale apomyoglobin, as induced by acid pH and by the addition of guanidine-HCI.

We describe a multidimensional spectrometer that is capable of (nearly) simultaneous measurement of circular dichroism, steady-state fluorescence, and absorbance values on the same sample in a standard 1 x 1 cm cuvette. With a computer controlled thermoelectric cell holder, this instrument is capable of measuring the above types of spectral data at various wavelengths as a function of temperature. We have developed software to control the various acquisition functions and to convert the data files to a format appropriate for use with the nonlinear least squares program, NONLIN (Johnson and Frasier, 1985). We have tested various features of this instrument and we have applied this instrument and data analysis procedure to study the thermal unfolding of ribonuclease A, under conditions were the unfolding of this protein involves an intermediate state.