Information about structural changes of protein molecules can be derived from optical measurements such as absorption, rotation, fluorescence, Raman scattering, etc. On the other hand, another unique optical method has recently been developed: photoacoustic spectroscopy (PAS).' It is not concerned with the radiative but with the nonradiative deexcitation of a sample. Now PAS is widely applied, but few measurements have been reported on the enzyme protein. The reason is that it is difficult to make aqueous solution without any gas bubbles and to get an intense light source in the violet where absorption spectra of protein molecules exist.
In the present experiment we performed photoacoustic studies about the denaturation of lysozyme. Thermal denaturation is a conformational phase transition accompanied by the unfolding of an antiparallel ,%structure. It is known also that various organic solvents act as a denaturant material on lysozyme. Upon denaturation by heat or chemicals, the highly organized globular configuration is destroyed and transformed into a random-coil state of chain molecules. Internal amino-acid residues are exposed outside, while hydrophobic bonds play a role in maintaining the native conformation of proteins. On reversal of the denaturant parameter, complete renaturation of the protein to the native state is achieved. We examined the application of PAS to studying these processes, comparing it with the absorption measurements.
Aqueous solution of lysozyme was contained in a transparent cell, carefully removing the gas bubbles in solution and on the cell wall. The light source of PAS was the third harmonic of a pulsed Nd:YAG laser with wavelength 355 nm and pulse duration 20 nsec. This wavelength is close to the point of absorption of the important amino-acid residue, tryptophan; PAS study of the conformational change was made a t this wavelength only. By using a pulsed light, we expected to reduce the influence of bubbles on the signal to the minimum possible. A piezoelectric solid transducer (lead zirconate titanate, PZT) was attached to the cell. Optical absorption measurements were performed with the usual conventional spectrometer. The spectrum for solution of 2 wt % lysozyme in water rose below 300 nm, a value known from tryptophan residues. However, there is also a small absorption taiI around 355 nm with the absorption coefficient, cm-I; this absorption gave a sufficient magnitude of the PAS signal. Excitation-intensity dependence of PAS was checked; it was slightly nonlinear at the higher-energy region (230 mJ/pulse), which would be due to two-photon absorption. Therefore, we made the input intensity weak enough in the linear region ( 2 5 mJ/pulse).