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Galectins pp 51-62 | Cite as

Alkylation of Galectin-1 with Iodoacetamide and Mass Spectrometric Mapping of the Sites of Incorporation

  • Sean R. Stowell
  • Connie M. Arthur
  • Richard D. Cummings
  • Christa L. FeasleyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1207)

Abstract

Galectins can display unique sensitivity to oxidative changes that result in significant conformational alterations that prevent carbohydrate recognition. While a variety of approaches can be utilized to prevent galectin oxidation, several of these require inclusion of reducing agents that not only prevent galectins from undergoing oxidative inactivation, but can also interfere with normal redox potentials required for fundamental cellular processes. To overcome limitations associated with placing cells in an artificial reducing environment, cysteine residues on galectins can be directly alkylated with iodoacetamide to form a stable thioether adduct that is resistant to further modification. Iodoacetamide alkylated galectin remains stable over prolonged periods of time and retains the carbohydrate binding and biological activities of the native protein. As a result, this approach allows examination of the biological roles of a stabilized form of galectin-1 without introducing the confounding variables that can occur when typical soluble reducing agents are employed.

Key words

Alkylation Galectin Mass spectrometry Oxidation Reducing agents 

Notes

Acknowledgments

This work was supported in part by grants from the National Blood Foundation, American Society of Hematology and Hemophilia of Georgia to S.R.S.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Sean R. Stowell
    • 1
  • Connie M. Arthur
    • 1
  • Richard D. Cummings
    • 2
  • Christa L. Feasley
    • 3
    Email author
  1. 1.Center for Transfusion and Cellular Therapies, Department of Pathology and Laboratory MedicineEmory University School of MedicineAtlantaUSA
  2. 2.Department of BiochemistryEmory University School of MedicineAtlantaUSA
  3. 3.Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical GlycobiologyUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA

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