Why tryptophan hydroxylase is difficult to purify: a reactive oxygen-derived species-mediated phenomenon that may be implicated in human pathology.
1. Attempts and apparently successful procedures to obtain reasonable quantities of electrophoretically homogenous mammalian brain-derived tryptophan hydroxylase, (TPH), have been described, starting in the early 1970s. This work has been carried out with the primary objective to obtain specific antisera to this enzyme to map out serotonergic pathways in the nervous system. 2. By using a multitude of techniques, antisera have indeed been fabricated and employed. However, it is doubtful if pure, native TPH has ever been produced. Indeed, there is strong evidence that more than one isoform of TPH exists in the rat brain. Thus, these antisera are probably directed against TPH-derived polypeptides and not the holoenzyme(s). 3. The difficulty in the purification of TPH lies not only in its subjectivity to proteolysis, but more importantly in its probable capacity to produce superoxide leading to hydrogen perioxide formation. This, in turn, may undergo Fenton chemistry with iron at the active site of the protein to produce hydroxyl radicals that directly attack and destroy the enzyme molecule. Evidence for such a mechanism is presented together with possible protocols that might be used to produce pure stable holo TPH(s). 4. It is hypothesized that similar oxidative events may take place in vivo under certain conditions leading to pathological results. Strategies to block these events are suggested.
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Centre de Neurochimie, Strasbourg, France.
D000818:Animals; D001921:Brain / Q000201:enzymology* / Q000378:metabolism; D005609:Free Radicals / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014365:Tryptophan Hydroxylase / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*
D005609:Free Radicals; D007527:Isoenzymes; D017382:Reactive Oxygen Species; D006899:Mixed Function Oxygenases; D014365:Tryptophan Hydroxylase