In situ analysis of proteins at high temperatures mediated by capillary-flow hydrothermal UV-vis spectrophotometer with a water-soluble chromogenic reagent.

In situ monitoring of quantities, interactions, and conformations of proteins is essential for the study of biochemistry under hydrothermal environments and the analysis of hyperthermophilic organisms in natural hydrothermal systems on Earth. We have investigated the potential of a capillary-flow hydrothermal UV-vis spectrophotometer (CHUS) for performing in situ measurements of proteins and determining their behavior at extremely high temperatures, in combination with a chromogenic reagents probe, which interacts with the proteins. The spectral shift obtained using a combination of water-soluble porphyrin (TPPS) and bovine serum albumin (BSA) was the best among the spectral shifts obtained using different combinations of chromogenic reagents and proteins. The association behavior of TPPS with BSA was investigated in detail using CHUS at temperatures up to 175 degrees C and the association constant (K(ass)) of TPPS with BSA was successfully determined at temperatures up to 100 degrees C. The lnK(ass) values were inversely proportional to the T(-1) values in the temperature range 50-100 degrees C. These analyses showed for the first time that the decrease of association of TPPS with BSA is due to the conformational change, fragmentation, and/or denaturing of BSA rather than the decrease of the hydrophobic association between TPPS and BSA. This study conclusively demonstrates the usability of the CHUS system with a chromogenic reagent as an in situ detection and measurement system for thermostable proteins at extremely high temperatures.