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Journal of Dairy Science Vol. 73 No. 9 2287-2291
© 1990 by American Dairy Science Association ®
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Confirmation of a Blocked Amino Terminus of Sulfhydryl Oxidase

Violeta G. Janolino 1, Sara J. Morrison-Rowe 1, and Harold E. Swaisgood 1

1 Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh 27695-7624

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)-kDa band 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 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.

Key Words: sulfhydryl oxidase • blocked amino terminus • radiolabeled active site

Submitted on January 5, 1990
Accepted on March 29, 1990






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Copyright © 1990 by the American Dairy Science Association ®.