Metal ligand-induced alterations in the surface structures of lactoferrin and transferrin probed by interaction with immobilized copper(II) ions.
We have evaluated immobilized Cu(II) ions as a potential site-directed molecular probe to monitor ligand-induced alterations in protein surface structures. Metal ion-induced alterations in the surface structures of different lactoferrins (human and porcine), transferrins (human and rabbit), and ovotransferrin (chicken) were examined. Although these 78,000-dalton glycoproteins are related gene products with similar overall structure and function, they differ greatly in the number and distribution of surface-exposed electron-donor groups thought to interact with Cu(II) ions. Each of these proteins interacted with immobilized Cu(II) ions through sites which are distinct from the two specific high affinity metal binding sites identified for iron. In both the presence and absence of bound iron, transferrins interacted more strongly with the immobilized Cu(II) ions than did lactoferrins; ovotransferrin interacted only weakly. Although iron binding increased the affinities of lactoferrins for immobilized Cu(II), iron binding decreased the affinities of transferrins and ovotransferrin for immobilized Cu(II) ions. Iron-saturated and iron-free lactoferrins were resolved by pH gradient elution, but only in the presence of 3 M urea; they were not resolved by imidazole affinity elution. Conversely, the iron-saturated and iron-free forms of transferrin were only separated by imidazole affinity elution. Urea did not influence the resolution of apo and holo ovotransferrins by imidazole. The differential effects of urea and imidazole suggest the participation of different types of surface electron-donor groups. The progressive site-specific modification of surface-exposed histidyl residues by carboxyethylation revealed several lactoferrin forms of intermediate affinity for immobilized iminodiacetate-Cu(II) ions. In summary, independent of species, the affinity for immobilized Cu(II) ions increased as follows: iron-saturated ovotransferrin less than metal-free ovotransferrin less than apolactoferrin less than hololactoferrin much less than diferric or holotransferrin less than monoferric transferrin less than apotransferrin. We have demonstrated the use of immobilized Cu(II) ions to distinguish and to monitor ligand-induced alterations in protein surface structure. The results are discussed in relation to protein surface-exposed areas of electron-donor groups.
Hutchens
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USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030.
Yip
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D000818:Animals; D002413:Cations, Divalent; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000737:chemistry*; D006801:Humans; D007781:Lactoferrin / Q000737:chemistry*; D008024:Ligands; D008670:Metals / Q000737:chemistry*; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet; D014168:Transferrin / Q000737:chemistry*
D002413:Cations, Divalent; D008024:Ligands; D008670:Metals; D014168:Transferrin; D003300:Copper; D007781:Lactoferrin