Specific detection of quinoproteins by redox-cycling staining.
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.
Laboratory of Human Biochemistry, Children's Hospital, Boston, Massachusetts 02115.
M A
MA
Paz
R
R
Flückiger
A
A
Boak
H M
HM
Kagan
P M
PM
Gallop
D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005998:Glycine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009580:Nitroblue Tetrazolium; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry*; D011809:Quinones / Q000737:chemistry*; D013194:Staining and Labeling
D011506:Proteins; D011809:Quinones; D009580:Nitroblue Tetrazolium; D005998:Glycine