Human histidine-rich glycoprotein: simultaneous purification with antithrombin III and characterization of its gross structure.
The simple and simultaneous purification of histidine-rich glycoprotein (HRG) and antithrombin III (AT III) from human plasma and gross structural characterization of HRG have been performed. The purification method consists of two chromatographic procedures using heparin-agarose and DEAE-Sephadex. The yields of HRG and AT III were 22 mg and 70 mg, respectively, from 1 liter of plasma. The purified HRG is a single-chain polypeptide with a molecular weight (Mr) of 75,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, indicating it was the native form of this protein. Cyanogen bromide cleavage of HRG, followed by analysis of the amino acid composition and determination of the amino-terminal sequence of each purified cyanogen bromide fragment established that the gross structure of HRG consisted of three cyanogen bromide fragments; an amino-terminal CN-50 kDa fragment (Mr 50,000) and a carboxy-terminal small fragment of eight amino acids, and a CN-30 kDa fragment (Mr 30,000) between them. As to the amino acid composition of the CN-30 kDa fragment, it had an unusually high content of histidine (25 mol%), suggesting the presence of a histidine-rich region(s) in the carboxy-terminal half of the molecule. These results together with our previous results (Koide, T., Odani, S., & Ono, T. (1982) FEBS Lett. 141, 222-224) and those of Morgan (Morgan, W.T. (1985) Biochemistry 24, 1496-1501) imply that HRG is composed of at least two domains with distinct functional properties; i.e. an amino-terminal domain with heparin-binding ability and a carboxy-terminal domain with heme- and divalent metal-binding abilities.
D000596:Amino Acids / Q000032:analysis; D000645:Ammonium Sulfate; D000990:Antithrombin III / Q000302:isolation & purification*; D002847:Chromatography, Agarose; D002848:Chromatography, DEAE-Cellulose; D003488:Cyanogen Bromide; D006801:Humans; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D000099053:Histidine-Rich Glycoprotein
D000596:Amino Acids; D010455:Peptides; D011506:Proteins; D000099053:Histidine-Rich Glycoprotein; D000990:Antithrombin III; D003488:Cyanogen Bromide; D000645:Ammonium Sulfate