Abstract
Low temperature (T less than or equal to 20 K) EPR measurements have revealed the presence of a heretofore undetected signal in uteroferrin, a purple protein bearing a single iron atom per molecule. All three of its principal g values (1.923, 1.738, and 1.583) lie well below the free electron value of 2.0023. Magnetic susceptibility data from 2-77 K confirm that the novel EPR spectrum arises from a paramagnetic center with a single unpaired electron spin. Quantitative correlation of the EPR, susceptibility, and optical data point to chromophoric iron as the source of the rhombic EPR spectrum. Furthermore, close agreement between the concentration of iron and the integrated intensity of the rhombic EPR signal show that the iron in the paramagnetic center is mononuclear. Reduction of the protein to its pink form leaves the rhombic signal essentially unaltered. The previously reported g' = 4.3 EPR signal accounts for only a small fraction of the total iron in the protein and undoubtedly arises from adventitious iron. Collectively, these results strongly suggest that uteroferrin represents a new class of low spin iron proteins.
Highlights
Low temperature(Ts20 K) EPR measuremenths ave revealed the presence of a heretofore undetected signal in uteroferrin, a purple protein bearing a single iron atom per molecule
Magnetic susceptibilitydata from 2-77 K confirm that the novel EPR spectrumarises from a paramagnetic center with a single unpaired electron spin
Close agreementbetween the concentration of iron and the integrated intensity of the rhombicEPR signal show that the ironin the paramagnetic center is mononuclear
Summary
Preparation of Uteroferrin-Uteroferrin was isolated according to published procedures [5, 11] from secretions that accumulate in the uteri of progesterone-treated sows. The first derivative EPR spectrum was stored in a Fabri-Tek model 1072signal averager and integrated to give the EPRabsorption short of the totaliron content for all three samples. Sample 111, a freshly prepared sample that suffered the least manipulation prior to these EPR studies, showed the lowest level of the g’ = 4.3 signal This result squareswell with optical data showing that thissample was the purest of the three,Le. A280/Ax5 = 13.0. 1.74EPR signal was determined similarlyexcept that a M solution of copper-EDTA in 2 M sodium perchlorate replaced diferric transferrin as the standard. In this case the dependence of transition. All spectra used for quantitative work were recorded at nonsaturating power levels.At a given temperature suitablepower levelswere determined by plotting the height of a prominent spectral feature (e.g.the eeak-to-peak height of the g, resonance of purple uteroferrin) versus JP,where P is the microwave power
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