Metal-catalyzed oxidation was used to identify metal-binding 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 >> (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.
(c)2001 Elsevier Science.