Khossravi M, Borchardt R T
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence 66047, USA.
Pharm Res. 1998 Jul;15(7):1096-102. doi: 10.1023/a:1011946631197.
To elucidate the nature of the reactive oxygen species (i.e., superoxide anion radical, hydroxyl radical, and hydrogen peroxide) involved in the metal-catalyzed oxidation of histidine (His) in two model peptides.
The degradation of AcAla-His-ValNH2 (Ala-peptide) and AcCysNH2-S-S-AcCys-His-VaNH2 (Cys-peptide) was investigated at pH 5.3 and 7.4 in an ascorbate/cupric chloride/oxygen (ascorbate/ Cu(II)/O2) system, both in the absence and presence of selective scavengers (i.e., catalase, superoxide dismutase, mannitol, sodium formate, isopropanol, and thiourea) of the reactive oxygen species. All reactions were monitored by HPLC. The major degradation products were characterized by electrospray mass spectrometry.
The Cys-peptide was more stable than the Ala-peptide at pH 5.3 and 7.4. Both peptides displayed greater stability at pH 5.3 than at 7.4. At pH 5.3, 35 +/- 0.7% of the Cys-peptide and 18 +/- 1% of the Ala-peptide remained after 7 hours, whereas at pH 7.4, 16 +/- 3% of the Cys-peptide and 4 +/- 1% of the Ala-peptide remained. Catalase, thiourea, bicinchoninic acid, and ethylenediaminetetraacetate were effective at stabilizing both peptides toward oxidation, while superoxide dismutase, mannitol, isopropanol, and sodium formate were ineffective. The main degradation products of the Ala- and Cys-peptides at pH 7.4 appeared to be AcAla-2-oxo-His-ValNH2 and AcCysNH2-S-S-AcCys-2-oxo-His-ValNH2, respectively.
Hydrogen peroxide, Cu(I), and superoxide anion radical were deduced to be intermediates involved in the oxidation of the Ala- and Cys-peptides. Hydrogen peroxide degradation to secondary reactive oxygen species may have led to the oxidation of the peptides. The degradation of hydrogen peroxide by a Fenton-type reaction was speculated to form a complexed form of hydroxyl radical that reacts with the peptide before diffusion into the bulk solution.
阐明参与两种模型肽中组氨酸（His）金属催化氧化反应的活性氧种类（即超氧阴离子自由基、羟基自由基和过氧化氢）的性质。
在抗坏血酸盐/氯化铜/氧气（抗坏血酸盐/Cu(II)/O2）体系中，于pH 5.3和7.4条件下，研究了AcAla - His - ValNH2（丙氨酸肽）和AcCysNH2 - S - S - AcCys - His - VaNH2（半胱氨酸肽）的降解情况，反应中有无活性氧种类的选择性清除剂（即过氧化氢酶、超氧化物歧化酶、甘露醇、甲酸钠、异丙醇和硫脲）。所有反应通过高效液相色谱法监测。主要降解产物通过电喷雾质谱法进行表征。
在pH 5.3和7.4条件下，半胱氨酸肽比丙氨酸肽更稳定。两种肽在pH 5.3时比在pH 7.4时表现出更高的稳定性。在pH 5.3时，7小时后半胱氨酸肽有35±0.7%留存，丙氨酸肽有18±1%留存；而在pH 7.4时，半胱氨酸肽有16±3%留存，丙氨酸肽有4±1%留存。过氧化氢酶、硫脲、二辛可宁酸和乙二胺四乙酸在使两种肽稳定以抵抗氧化方面有效，而超氧化物歧化酶、甘露醇、异丙醇和甲酸钠无效。在pH 7.4时，丙氨酸肽和半胱氨酸肽的主要降解产物似乎分别是AcAla - 2 - 氧代 - His - ValNH2和AcCysNH2 - S - S - AcCys - 2 - 氧代 - His - ValNH2。
推断过氧化氢、Cu(I)和超氧阴离子自由基是参与丙氨酸肽和半胱氨酸肽氧化反应的中间体。过氧化氢降解为二级活性氧种类可能导致了肽的氧化。推测通过芬顿型反应降解过氧化氢会形成一种与肽反应的络合形式的羟基自由基，该自由基在扩散到本体溶液之前就与肽发生反应。
