Preparation of irreversibly sickled cell β-actin from normal red blood cell β-actin

A. Abraham, F. A. Bencsath, A. Shartava, D. G. Kakhniashvili, Steven Goodman

Research output: Contribution to journalArticle

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Abstract

We have previously demonstrated that an oxidative change, the formation of a disulfide bridge between two cysteine residues, in the membrane protein β-actin is primarily responsible for locking the irreversibly sickled red blood cells (ISCs) of sickle cell anemic patients into the sickle shape. To support studies on biological and chemical characterization of the oxidized β-actin and pharmacological research toward the reversal of the oxidation, we attempted to prepare oxidized β-actin from normal red blood cell (RBC) β-actin by a chemical reaction, expecting a product equivalent to that found in ISCs. 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB, or Ellman's reagent) was used for the oxidation. We proved the absence of accessible sulfhydryl groups in the oxidized product using liquid chromatography (LC) with both UV and fluorescence detection. Polymerization assays indicated that the chemically produced ISC actin demonstrated the same kinetics as ISC actin obtained from patients with sickle cell disease. The effect of the oxidation could be reversed by the use of the reducing agent tris(carboxyethyl)phosphine (TCEP).

Original languageEnglish (US)
Pages (from-to)292-296
Number of pages5
JournalBiochemistry
Volume41
Issue number1
DOIs
StatePublished - Jan 8 2002

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Actins
Blood
Erythrocytes
Cells
Dithionitrobenzoic Acid
phosphine
Oxidation
Nitrobenzoates
Reducing Agents
Liquid chromatography
Sickle Cell Anemia
Liquid Chromatography
Polymerization
Disulfides
Cysteine
Chemical reactions
Assays
Membrane Proteins
Fluorescence
Pharmacology

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Preparation of irreversibly sickled cell β-actin from normal red blood cell β-actin. / Abraham, A.; Bencsath, F. A.; Shartava, A.; Kakhniashvili, D. G.; Goodman, Steven.

In: Biochemistry, Vol. 41, No. 1, 08.01.2002, p. 292-296.

Research output: Contribution to journalArticle

Abraham, A, Bencsath, FA, Shartava, A, Kakhniashvili, DG & Goodman, S 2002, 'Preparation of irreversibly sickled cell β-actin from normal red blood cell β-actin', Biochemistry, vol. 41, no. 1, pp. 292-296. https://doi.org/10.1021/bi010685v
Abraham, A. ; Bencsath, F. A. ; Shartava, A. ; Kakhniashvili, D. G. ; Goodman, Steven. / Preparation of irreversibly sickled cell β-actin from normal red blood cell β-actin. In: Biochemistry. 2002 ; Vol. 41, No. 1. pp. 292-296.
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