F R E E R A D I C A L S I N M E L A N I N B . T . Al l e n
Department of Biochemistry, University of Oregon Medical School, Portland, U.S.A.
Free radicals, which have electrons with unpaired spins, such as occur in photochemical or oxidation processes or are trapped in polymers, can be detected and examined by the methods of electron spin resonance (e.s.r.). When placed in a magnetic field substances con- taining unpaired electrons absorb long-wave electromagnetic radia- tion. This is because the energy level of an unpaired electron is split by the magnetic field into two levels, and electromagnetic radiation can cause transitions between them. The levels differ in energy by gBH9 where Β is the ' Bohr magneton ', Η is the magnetic field strength, and g is the 'splitting factor', which has the value 2-0023 for free spin and somewhat different values for spins of electrons bound in molecules.
It can be shown theoretically that absorption can occur only when electrons in the higher level can lose energy by radiationless effects due to their environment; this is characterized by a 'spin-lattice relaxation time'. If this time is relatively long, as is usual for free radicals, increase in the radio-frequency magnetic field will produce 'saturation', shown by an apparent decrease of absorption and often by increase of 'line-width'. Other magnetic effects are observable for molecules, from electron magnetic dipoles and nuclear spins, but these seem to be negligible for melanin. By a careful study of level splitting, g-value and 'spin-spin' relaxation time, spin-lattice relaxa- tion time, line width and line shape, with saturation effects due to field strength, information can be obtained about the molecular environment of the electrons with unpaired spins.
Bl o i s et al (1964) have examined these signal parameters for both
natural squid melanin and artificial pigments. In all cases £-values of 2-0030 to 2-0048 were obtained at fields of 4-9 G, and spin-lattice relaxation times were long enough to allow of saturation at low fields.
As this affects the absorption intensity, changes of line height do not necessarily correlate with changes of unpaired spin electron con- centration. The work emphasizes the need for determination of all experimental parameters to derive reliable information.
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378 L I G H T A N D M E L A N I N S K I N P I G M E N T A T I O N
The effects of environmental change on the intensity of the e.s.r.
signal have been examined by Co p e et al (1963), Bl o i s et al (1964),
Ke r k u t et al (1962) and Co t z i a s et al (1964). Co p e has found that the water content affected only the dark signal while Bl o i s showed a rise and then fall of intensity on drying. Change of pH from 6-10 resulted in a ten-fold increase of signal intensity. No correction for saturation was made, and the results may be due to spin-lattice relaxation effects.
The temperature effect is consistent with Curie's Law down to about 4 ° K . If the pigment is stored at excess of 8o° C prior to examination, an increased signal intensity could be obtained.
According to Co p e, the signal shows some dependence on oxygen tension, but Bl o i s suggests oxygen cannot react with the unpaired spin sites. The presence of cations is found to reduce spin-lattice relaxation time. Considerable differences were observed when the metals were removed from the calcium-magnesium melanin com- pound. Co t z i a s demonstrated a distinct correlation between pig- mentation and manganese content.
An increase in signal intensity when melanin is irradiated was reported by Ma s o n et al (i960), and denied by Ke r k u t, but no temperature measurements were given. Co p e has shown a significant increase with controlled conditions. Protein results in a decrease of free radical formation in u.v. irradiated melanin, according to Ke r k u t ,
due to interaction between the separate species of free radicals.
The e.s.r. signal may originate from free radicals, conduction electrons, transition metals or singlet and triplet states. Only the first two are considered. Lo n g u e t- Hi g g i n s (i960) and Pu l l m a n and
Pu l l m a n (1961), using molecular orbital theory, suggest the existence of a low-lying, unfilled molecular orbital in polymers. If this theory were extended to monomer units, a one-electron trap could be postulated. E.s.r. signals may be observed from either conduction electrons or trapped conduction electrons. The close accordance with the Curie Law and the negative results of Bl o i s with pH change support neither case. The increase in both light and dark signal above pH 6 shown by Co p e, however, might well agree the existence of unpaired electrons, trapped on single monomer units of an oxidised quinonoid polymer.
The temperature dependence and high £-values are consistent with the signal source being free radical in origin. The absence of hyperfine structure is explained by Bl o i s as being the net product of a number of varying hyperfine spectra. The inability to rotate when trapped in the
F R E E R A D I C A L S I N M E L A N I N 379
polymer would also explain this. Al l e n and Va n n e s t e (1964) showed no difference in absorption lines with the brown pigment formed by anaerobic oxidation with eerie ammonium sulphate of chloro deriva- tives of phenol although these would be expected if hyperfines were smeared out. The absorption curve reverted from Gaussian to an intermediate with the Lorentzian shape by spin-spin dipolar inter- action, and the absence of hyperfine structure indicates that trapped radicals occur as closely adjacent pairs.
The biological function of melanin would seem best elucidated from the irradiation signals. All parameters of these signals must be taken into account and signal height must not be overemphasized. The signal observed by Co p e was identical for the light and the dark situation. Whether it is permanent or long lived in certain circum- stances is not clear.
R E F E R E N C E S
Bl o i s M . S . , Za h l a n A.B., Ma l i n g J.E. (1964) Biophys. J. (in press).
Cope F.W., Sever R.J., Po l i s B.D. (1963) Arch. Biochem. Biophys. 1 0 0 , 171.
Co t z i a s G . C . , Pa p a v a s i l i o u P . S . , Mi l l e r S . T . (1964) Nature, Lond. 2 0 1 , 1228.
Ke r k u t G . A . , Ed w a r d s M . L . , Mu n d a y K . A . (1962) Life Sciences p. 129.
Lo n g u e t- Hi g g i n s H . C . (i960) Arch. Biochem. Biophys. 86, 231.
Ma s o n H.S., In g r a m D J . E . and Al l e n B . T . (i960) Arch. Biochem. Biophys.
86, 225.
Pu l l m a n A. and Pu l l m a n B . (1961) Biochem. Biophys. Acta 54, 384.
