The use of dithionite reduction to identify the essential tyrosine residue in the F1-ATPase from the thermophilic bacterium, PS3, that reacts with 7-chloro-4-nitrobenzofurazan.
When the F1-ATPase from the thermophilic bacterium, PS3, was inactivated by greater than 90% with 7-chloro-4-nitro[14C]benzofurazan ([14C]Nbf-Cl) at pH 7.4, 1.4 mol of [14C]Nbf were incorporated per mol of enzyme. After pepsin digestion of the labeled enzyme at pH 3.0, a single, major peak of radioactivity was resolved by reversed-phase high-performance liquid chromatography under acidic conditions were peptidyl Nbf-O-tyrosine is stable. This radioactive peak, designated RP-1, eluted with a retention time of 95 min. When the material in RP-1 was subjected to reversed-phase high-performance liquid chromatography under the same conditions after treatment with sodium dithionite, a single, major peak of radioactivity, designated RP-2, was resolved with a retention time of 52 min. Automatic Edman degradation of this material revealed that it has the amino acid sequence I-Y*-V-P-A-D-(D), where Y* presumably represents peptidyl [14C]Nbf-O-tyrosine. These results provide the basis for a facile method to purify peptides containing [14C]Nbf-O-tyrosine in which the labeled residues can be identified by amino acid sequence analysis using the Edman degradation.
Verburg
J G
JG
Yoshida
M
M
Allison
W S
WS
D009327:4-Chloro-7-nitrobenzofurazan / Q000378:metabolism*; D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D001665:Binding Sites / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D004227:Dithionite / Q000494:pharmacology*; D006358:Hot Temperature; D010069:Oxadiazoles / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013447:Sulfites / Q000494:pharmacology*; D014443:Tyrosine / Q000032:analysis*
D010069:Oxadiazoles; D013447:Sulfites; D004227:Dithionite; D014443:Tyrosine; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan