Abstract
Quinones and related quinonoid substances catalyze redox cycling at an alkaline pH in the presence of excess glycine as reductant. With nitroblue tetrazolium and oxygen present there is concomitant reduction of the tetrazolium to formazan. This property of quinonoid compounds is used for the specific staining of quinoproteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. The dopa-containing vitelline proteins and the 6-hydroxydopa-containing bovine serum amine oxidase are stained with the nitroblue tetrazolium/glycinate reagent. Also, the mammalian quinoproteins, diamine oxidase and lysyl oxidase, purported to contain pyrroloquinoline quinone, tested positive in this procedure. No quinonoid components were detected in three putative pyrroloquinoline quinone-containing quinoproteins, dopamine beta-hydroxylase, lipoxygenase, and peptidylglycine-amidating monoxygenase. Redox-cycling staining therefore confirms the presence of covalently bound quinones in the copper-dependent amine oxidases, but not in two putative quinoprotein oxygenases. Clarification of the biological significance of quinolation should be facilitated by identification of quinoproteins using this approach.
Highlights
Quinones and related quinonoid substances catalyze redox cycling at analkaline pH in the presence of excess glycineas reductant.With nitrobluetetrazolium and oxygen present there is concomitant reduction of the tetrazolium to formazan
Earlier evidence suggested that pyrroloquinoline quinone (PQQ) is the orthoquinone cofactor in the mammalian quinoproteins, lysyl oxidase from bovine aorta [3], and human placen(t4a), pig kidney diamine oxidase ( 5 ),bovine plasma amine oxidase [6, 7], bovine adrenal dopamine @-hydroxylase[8], dog liver choline dehydrogenase [9], pig kidney dopa decarboxylase, and possible in peptidylglycine-amidatingmonooxygenase [1]
We have found that PQQ oxidizes glycine at an alkaline pH but not valine
Summary
Quinones and related quinonoid substances catalyze redox cycling at analkaline pH in the presence of excess glycineas reductant.With nitrobluetetrazolium and oxygen present there is concomitant reduction of the tetrazolium to formazan. Earlier evidence suggested that PQQ is the orthoquinone cofactor in the mammalian quinoproteins, lysyl oxidase from bovine aorta [3], and human placen(t4a), pig kidney diamine oxidase ( 5 ) ,bovine plasma amine oxidase [6, 7], bovine adrenal dopamine @-hydroxylase[8], dog liver choline dehydrogenase [9], pig kidney dopa decarboxylase (lo), and possible in peptidylglycine-amidatingmonooxygenase [1]. We have used this reaction for the identification of PQQ-like substances and quinonoid compounds in biological fluids, quinoproteins, and quinopeptides [18].We report how this redox-cycling property of quinones can be used for the detection of quinoproteins after separation and electroblotting onto nitrocellulose.
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