Preparation of irreversibly sickled cell beta-actin from normal red blood cell beta-actin.
We have previously demonstrated that an oxidative change, the formation of a disulfide bridge between two cysteine residues, in the membrane protein beta-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 beta-actin and pharmacological research toward the reversal of the oxidation, we attempted to prepare oxidized beta-actin from normal red blood cell (RBC) beta-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).
Abraham
Ann
A
Mass Spectrometry and Protein Structure Laboratory, University of South Alabama, Mobile, Alabama 36688, USA.
Bencsath
F Aladar
FA
Shartava
Archil
A
Kakhniashvili
David G
DG
Goodman
Steven R
SR
D000199:Actins / Q000302:isolation & purification*; D000328:Adult; D000755:Anemia, Sickle Cell / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000737:chemistry*; D006801:Humans; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism
D000199:Actins; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; D014357:Trypsin