100-microsecond-resolved exafs technique for studying photolyzed hemoproteins
Huang, Huey W.
Master of Arts
This thesis describes a 1-jus-resolved extended x-ray absorption fine structre (EXAFS) technique for studying protein dynamics. Both the time-resolved EXAFS spectrometer and the protein samples are described in detail. About ten years ago synchrotron radiation from electron storage rings began to be used for research in condensed matter. As a result a new technique for studying local structures in non-crystalline system was developed i.e. the extended x-ray absorption fine structure. This technique has now become an important tool for the structural studies of biological systems, particularly metalloproteins. In recent years the development of wiggler and undulator magnets has increased the radiation intensity to a level that, according to photon statistics, it should be possible to measure the EXAFS of hemoproteins in their transient states. Such measurements would provide structural insight to the very difficult but important problem of protein dynamics. However the conventional method of EXAFS measurement is inefficient for time-resolved measurement. We have developed a new spectrometer to take full advantage of the intense radiation. The time resolution of our spectrometer is about 1 us; below that the measurement time would be too long. Myoglobin was chosen as our sample for its importance in protein biophysics. The complex of myoglobin and carbon monoxide (CO) can be dissociated by light (flash photolysis). Its recombination time ranges from microseconds to infinity depending on temperature. The time resolved EXAFS measurement of photolyzed carboxymyoglobin will reveal the structural changes of protein around the CO binding site during the recombination process. We made our sample in the form of a thin film so that it can meet different requirments of x-ray absorption and optical photolysis. A special procedure of sample making was developed, and a transient optical absorption spectrometer was built for testing the samples. All optical absorption properties of our samples are in agreement with known results. An actual time-resolved EXAFS experiment was performed in February, 1983. The result showed no change in EXAFS with time. This might be due to a failure in photolysis (we lacked an on-line transient optical absorption spectrometer). However, these results demonstrated the feasibility and reliability of our spectrometer.