1991 Fiscal Year Final Research Report Summary
Rapid measurement of oxygenin tissues utilijing palce flash photolysis
| Project/Area Number |
02557096
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
医学一般
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
ORII Yutaka Kyoto University,Fac.of Medicine, Associate Professor, 医学部, 助教授 (60028149)
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| Co-Investigator(Kenkyū-buntansha) |
NAGAMURA Toshihiko YUNISOKU Company of limited, President, 代表取締役
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| Project Period (FY) |
1990 – 1991
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| Keywords | tissure oxygen / rapid scan / pulse flash / laser flash / hemoglobin / myoglobin |
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| Research Abstract |
1.An ablolutely anaerobic flow flash apparatus was constructed by incorporating ballvalves made of ruby balls to switch the flow path of the sample solutions. Also pressurized nitrogen gas can be used to purge air oxygen from the contact of the piston head of the driving syringe to assure the anaerobiosis. These improvements allow to follow the spectral change of a sample solution in the observation cell in a time range from 1 ms to 50 min without being interfered from contamination of air oxygen. Using this apparatus the spectral changes upon dithionite reduction of cytochrome oxidase were followed in a time range from 1 ms to 30 min, based on which the spectral separation of two heme alpha moieties in this enzyme was achieved. The absolute absorption spectra thus derived can be used successfully to explain multiphasic absorbance changes at any selected wavelengths in the Soret region during the reduction process. These spectral data are fundamental to delineate the intricate function of this enzyme to convert the redox free energy into the proton driving force across the biological membrane.
2. In the proton pumping of cytochrome oxidase Cu_A has been assumed to play a crucial role. By using cytochrome oxidase from Nitrosomonas europaea which lacks Cu_A its reaction with oxygen was studied by using a flow flash apparatus. This Cu_A-deficient enzyme, however, behaves like a normal enzyme; the rate for the reaction with oxygen as well as for the intramolecular electron transfer is even higher than that of the normal enzyme. This finding has derived significant implications that Cu_A is not essential to the electron transfer process but assumed to keep and control the tertiary structure of the enzyme which is essential to the function of proton translocation.
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