Metal ion affinity adsorption of a Zn(II)-transport protein present in maternal plasma during lactation: structural characterization and identification as histidine-rich glycoprotein.
A high-affinity Zn(II)-binding protein has been purified to homogeneity (880-fold) from the plasma of lactating women by a single affinity adsorption step on columns of tris(carboxymethyl)ethylenediamine (TED)-agarose loaded with Zn(II) ions. Purity was evaluated by high-performance reverse-phase (phenyl) chromatography and by silver staining after SDS-polyacrylamide gradient gel electrophoresis. The mass of denatured Zn(II)-binding protein was estimated by SDS-polyacrylamide gradient gel electrophoresis to be 75 kDa under both reducing and nonreducing conditions; by matrix-assisted uv laser desorption time-of-flight mass spectrometry the purified protein mass was determined to be 66 kDa. The amino acid composition revealed a high content of His (13 mol%) and Pro (12 mol%). N-terminal amino acid sequence analysis (50 residues) identified the purified protein as histidine-rich glycoprotein (HRG). Immunoblots demonstrated the absence of fragments in the purified product. An enzyme-linked immunosorbent assay was developed; a 75% recovery of intact HRG from the immobilized Zn(II) ion affinity column was documented. The circular dichroism spectra for the purified human HRG in the far uv (260-178 nm) were similar to those published for human and rabbit serum HRG. These results demonstrate that TED-immobilized Zn(II) ions can be used as a new and efficient method for the isolation of structurally intact human plasma HRG.
Yip
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USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston 77030.
Hutchens
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002352:Carrier Proteins / Q000097:blood* / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007774:Lactation / Q000097:blood; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D015032:Zinc / Q000097:blood*; D000099053:Histidine-Rich Glycoprotein
D000596:Amino Acids; D002352:Carrier Proteins; D011506:Proteins; D000099053:Histidine-Rich Glycoprotein; C017800:zinc-binding protein; D015032:Zinc