PHOTOACOUSTIC ANALYSIS OF PROTEINS - VOLUMETRIC SIGNALS AND FLUORESCENCE QUANTUM YIELDS

A series of proteins has been examined using time-resolved, pulsed-las er Volumetric photoacoustic spectroscopy, Photoacoustic waveforms were collected to measure heat release for calculation of fluorescence qua ntum yields, and to explore the possibility of photoinduced nonthermal volume changes occurring in these protein samples, The proteins studi ed were the green fluorescent protein (GFP); intestinal fatty acid bin ding protein (IFABP), and adipocyte lipid-binding protein (ALBP), each labeled noncovalently with 1-anilinonaphthalene-8-sulfonate (1,8-ANS) and covalently with 6-acryloyl-2-(dimethylamino)naphthalene (acryloda n); and acrylodan-labeled IFABP and ALBP with added oleic acid. Of thi s group of proteins, only the ALBP labeled with I,8-ANS showed signifi cant nonthermal volume changes at the beta = 0 temperature (similar to 3.8 degrees C) for the buffer used (10 mM Tris-HCl, pH 7.5) (beta is the thermal cubic volumetric expansion coefficient), For all of the pr oteins except for acrylodan-labeled IFABP, the fluorescence quantum yi elds calculated assuming simple energy conservation were anomalously h igh, i.e., the apparent heat signals were lower than those predicted f rom independent fluorescence measurements, The consistent anomalies su ggest that the low photoacoustic signals may be characteristic of fluo rophores buried in proteins, and thar photoacoustic signals derive in part from the microenvironment of the absorbing chromophore.