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| TITLE: | Magnetic Spin Effects in Radical Enzymatic Reactions | ||
| Principal Investigator |
Charles B. Grissom | University of Utah | |
| Health Relevance |
Other: Biophysics of Cellular Function | ||
| Research Categories |
Cellular Function | Biophysics Cell/Field | Cell signaling |
| FY95 Funds | R01ES05728 $ 134,034 | Start Date 4/15/93 | End Date 3/31/96 Rationale and |
| Rationale and Summary |
Although there have been several reports in the scientific literature suggesting a link between
exposure to electric and magnetic fields and the development of cancer, there are few laboratory
based studies that offer a possible mechanism. The objective of this work is to study the influence
of static (DC) magnetic fields on enzymatic reactions with radical (unpaired electron) intermediates
in order to understand how environmental magnetic fields might influence biological processes
through changes in radical pair recombination. A secondary goal of this work is to develop new
tools to study the catalytic mechanism of enzymes with radical intermediates.
Only biochemical reactions that involve more than one unpaired electron will be affected by a magnetic field. Most enzymes do not involve radical intermediates and should be unaffected by a change in magnetic field. However, more than 50 enzymes are believed to generate free radical intermediates during catalysis and may be subject to the influence of external magnetic fields. During the past two years, the Grissom research group has shown that the activity of the B12- dependent enzyme ethanolamine ammonia lyase changes with magnetic field. This is the first magnetic field effect in an enzyme-catalyzed reaction with known radical pair intermediates. |
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| Experimental Design and Exposure Conditions |
The effect of an external magnetic field on enzymatic reaction rates can be determined in the same way classical enzyme kinetic parameters are determined. Enzymes with chromogenic substrates or products can be followed spectrophotometrically. If no convenient chromophore is available for direct measurement of enzyme rates, then a fixed-time assay with HPLC or radionuclide detection of products or residual substrate can be used. A variety of photolysis light sources, monochromators, and filters are available in our laboratory. We have two electromagnet systems relevant to this work. (System 1): A single-beam UV/Vis spectrophotometer with an electromagnet for thermostatted steady-state kinetic measurements in the range 0-2500 gauss. (System 2): A rapid-scanning stopped-flow spectrophotometer with an electromagnet for rapid kinetic measurements also in the range of 0-6500 gauss. This instrument has some very desirable characteristics for our work with cobalamins and heme enzymes: it acquires an absorbance spectrum at a rate of 1,000 Hz with photomultiplier sensitivity. This is accomplished with a scanning wheel and double monochromator design that is unique to the OLIS, Inc. instrument. This system is equipped with an electromagnet and gaussmeter. | ||
| Quality Assurance Measures |
The spectrophotometers used in these experiments are shielded from the electromagnet, and we
have tested these systems for adventitious magnetic field effects by looking for the influence of the
magnetic field on enzymes without radical pair intermediates. No magnetic field effect was
observed on these "control" enzymes.
Furthermore, our magnetic field effect results with B12 ethanolamine ammonia lyase have been repeated and verified by Professor Ruma Banerjee in the Department of Biological Chemistry at the University of Nebraska. A manuscript detailing this replication is in preparation. |
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| Results and Discussion |
Magnetic field effects on radical pair recombination rates tend to exhibit "window" effects, such
that higher fields do not always produce correspondingly larger effects. Because the fields are
static, a simple laboratory electromagnet is adequate to achieve stable magnetic fields up to 10,000
gauss. The effect of a magnetic field on an enzymatic reaction can be observed by a change in the
steady-state kinetic parameters, as well as by a change in the stopped-flow kinetic parameters (two
different measurement techniques and two different instruments). The same magnetic field effect
can be seen on the rate of photolysis of the vitamin B12 cofactor (without enzyme).
The rate of another B12 enzyme, methylmalonyl CoA mutase (from humans), is not altered by a magnetic field, in spite of many similarities between these reactions. Experiments are underway to understand this different response to magnetic fields. Experiments are also being carried out with the heme enzymes horseradish peroxidase and cytochrome P-450. The rate of horseradish peroxidase increases by 20% at fields as low as 10 gauss. Other heme enzymes are expected to exhibit a magnetic field dependence at environmentally relevant magnetic fields of 10 gauss and below. |
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| Recent Publications |
Miller, J. R.; Edmondson, D. E.; Grissom, C. B. "Mechanistic Probes of Monoamine Oxidase B
Catalysis: Rapid-Scan Stopped-Flow and Magnetic Field Independence of the Reductive Half
Reaction" J. Am. Chem. Soc. 1995, 117, 7830-7831.
Harkins, T. T.; Grissom, C. B. "The Magnetic Field Dependent Step in B12 Ethanolamine Ammonia Lyase is Radical-Pair Recombination." J. Am. Chem. Soc. 1995, 117, 566-567. Grissom, C. B. "Magnetic Field Effects in Biology: A Survey of Possible Mechanisms with Emphasis on Radical-Pair Recombination." Chem. Rev. 1995, 95, 3-24. |
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