Confirmation of a blocked amino terminus of sulfhydryl oxidase

Journal Article · Sat Sep 01 00:00:00 EDT 1990 · Journal of Dairy Science; (USA)

DOI:https://doi.org/10.3168/jds.S0022-0302(90)78909-6· OSTI ID:5962352

Janolino, V G; Morrison-Rowe, S J; Swaisgood, H E ^[1]

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1. North Carolina State Univ., Raleigh (USA)

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 ({sup 14}C)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 ({plus minus} 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.

• OSTI ID: 5962352
• Journal Information: Journal of Dairy Science; (USA), Journal Name: Journal of Dairy Science; (USA) Vol. 73:9; ISSN 0022-0302; ISSN JDSCA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
CARBON 14 COMPOUNDS
DNA SEQUENCING
ELECTROPHORESIS
ENZYMES
HALOGEN COMPOUNDS
INACTIVATION
IODINE COMPOUNDS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LABELLING
METALLOPROTEINS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXIDOREDUCTASES
PROTEINS
STRUCTURAL CHEMICAL ANALYSIS
THIOLS
TRACER TECHNIQUES