Characterization of the Metal-Binding Site of Bovine Growth Hormone through Site-Specific Metal-Catalyzed Oxidation and High-Performance Liquid Chromatography–Tandem Mass Spectrometry

✍ Scribed by Susan W. Hovorka; Todd D. Williams; Christian Schöneich

• Publisher: Elsevier Science
• Year: 2002
• Tongue: English
• Weight: 98 KB
• Volume: 300
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2001.5447

✦ Synopsis

Metal-catalyzed oxidation was used to identify metalbinding His residues in bovine growth hormone (bGH), which has not been characterized well crystallographically due to a high propensity of bGH to aggregate. bGH was exposed to Cu 2؉ and ascorbate (ascorbate/Cu 2؉ /O 2 ). 2-Oxo-His formation was identified by HPLC-tandem mass spectrometry (MS/MS) analysis of a tryptic digest. Two 2-oxo-His-containing fragments were detected, T2(O) (MH 2 2؉ ‫؍‬ 748.8) and T20(O) (MH ؉ ‫؍‬ 528.3), both masses corresponding to the addition of only one oxygen atom (؉16 amu) to the respective native fragments, T2 and T20. T2 contains 20 His and 22 His, and T20 contains 170 His. Quantitative HPLC-MS/MS analysis shows the following order of reactivity: 170 His ӷ 22 His > 20 His. Solvent-accessible surface area calculations determined 22 His and 170 His to be 26 and 35% solvent exposed, respectively, while 20 His is 65% solvent exposed. The presence of an analogous metal-binding site in human growth hormone, which is located in the hydrophobic core, and our experimental finding that oxidation was greatest for 22 His and 170 His in bGH suggests that 22 His and 170 His of bGH participate in metal binding. This result is supported by a previously predicted tertiary structure of bGH and compared with the location of metal-binding His residues of human growth hormone.