期刊
ANALYTICAL BIOCHEMISTRY
卷 290, 期 1, 页码 138-145出版社
ACADEMIC PRESS INC
DOI: 10.1006/abio.2000.4958
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资金
- NHLBI NIH HHS [HL-65608, HL-38324, HL-63744] Funding Source: Medline
- NIA NIH HHS [AG00835] Funding Source: Medline
Hydroxylation of L-phenylalanine (Phe) by hydroxyl radical ((OH)-O-.) yields 4-, 3-, and 2-hydroxyl-Phe (para-, meta-, and ortho-tyrosine, respectively), Phe derivative measurements have been employed to detect (OH)-O-. formation in cells and tissues, however, the specificity of this assay is limited since Phe derivatives also arise from intracellular Phe hydroxylase. D-Phe, the D-type enantiomer, is not hydroxylated by Phe hydroxylase. We evaluate whether D-Phe reacts with (OH)-O-. as well as L-Phe, providing a more reliable probe for (OH)-O-. generation in biological systems. With (OH)-O-. generated by a Fenton reaction or xanthine oxidase, D- and L-Phe equally gave rise to p, m, o-tyr and this could be prevented by (OH)-O-. scavengers. Resting human neutrophils (PMNs) markedly converted L-Phe to p-tyr, through non-oxidant-mediated reactions, whereas D-Phe was unaffected. In contrast, when PMNs mere stimulated in the presence of redox cycling iron the (OH)-O-. formed resulted in more significant rise of p-tyr from D-Phe (9.4-fold) than L-Phe (3.6-fold) due to the significant background formation of p-tyr from L-Phe. Together, these data indicated that D- and L-Phe were equally hydroxylated by (OH)-O-.. Using D-Phe instead of L-Phe can eliminate the formation of Phe derivatives from Phe hydroxylase and achieve more specific, sensitive measurement of (OH)-O-. in biological systems. (C) 2001 Academic Press.
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