Confirmation of a blocked amino terminus of sulfhydryl oxidase.
The isolation of sulfhydryl oxidase from bovine milk in a suitably pure form for sequencing was carried out by transient covalent affinity chromatography of diafiltered whey using cysteinylsuccinamidopropyl-glass as matrix. The glutathione-eluted proteins were separated by SDS-PAGE. By radiolabeling the affinity chromatography-purified enzyme with [14C]iodoacetate before subjecting to SDS-PAGE, the sulfhydryl oxidase band was identified, because sulfhydryl oxidase is known to be inactivated by alkylation of one sulfhydryl group per mole. The results confirmed that sulfhydryl oxidase corresponds to the 85 (+/- 5)-k-Daband observed on SDS-PAGE. The protein band corresponding to radiolabeled sulfhydryl oxidase was recovered from SDS-PAGE gels by electrophoretic elution and by electroblotting on polyvinylidene difluoride membrane and polyvinylidene difluoride membrane and subjected to gas phase sequencing. Precautions were taken during electrophoretic elution to prevent reactions that result in N-terminal blocking. Both methods of protein recovery yielded negative results when subjected to sequence analysis indicating that the N-terminus of sulfhydryl oxidase is blocked.
Janolino
V G
VG
Department of Food Science, North Carolina State University, Raleigh 27695-7624.
Morrison-Rowe
S J
SJ
Swaisgood
H E
HE
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D008892:Milk / Q000201:enzymology*; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification; D011145:Polyvinyls; D050862:Oxidoreductases Acting on Sulfur Group Donors; D005465:Fluorocarbon Polymers
D010088:Oxidoreductases; D011145:Polyvinyls; D050862:Oxidoreductases Acting on Sulfur Group Donors; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers