PERIODATE MODIFICATION OF HUMAN SERUM TRANSFERRIN FE(III)-BINDING SITES - INHIBITION OF CARBONATE INSERTION INTO FE(III)-CHELATOR-TRANSFERRIN AND CU(II)-CHELATOR-TRANSFERRIN TERNARY COMPLEXES

Periodate modification of human serum transferrin produces a species t hat binds Fe(III) weakly at pH 7.4 contrary to previous reports that F e(III)-binding activity is completely lost, Ternary complexes of perio date-modified transferrin and either Fe(III) with nitrilotriacetate (N TA), oxalate, citrate, or EDTA, or of Cu(II) with oxalate could be for med. Peak wavelength maxima of these spectral bands are identical to t hose reported for native transferrin in the absence of bicarbonate. No carbonate ternary complexes of periodate-modified transferrin with Fe (III), Al(III), Cu(II), or Zn(II) could be formed. Conditional (Fe(NTA )) binding constants (log K) for C- and N-terminal modified sites are 7.33 and 7.54, respectively, The respective extinction coefficients at 470 nm are decreased 45% compared with the native protein. The electr on paramagnetic resonance spectrum of the complex closely resembles th at of the Fe(III) NTA ternary complex formed with native transferrin i n the absence of bicarbonate. Anions, including bicarbonate, at high c oncentrations destabilize formation of this Fe(III)NTA ternary complex , while Fe(III) chelators readily remove the bound Fe(III), Bicarbonat e, sulfate, and pyrophosphate still bind to the modified binding sites in the absence of metal although with slightly lower affinity and wit h lower molar difference absorptivities, Results are interpreted as an inhibition of a crucial protein conformational change by an intramole cular cross-link, preventing formation of the particularly stable meta l carbonate ternary complex hom the less stable metal-chelate ternary complex, The method can be used to produce monosited transferrins.