Preparation of Irreversibly Sickled Cell -Actin from Normal Red Blood Cell -Actin †,‡ Ann Abraham, § F. Aladar Bencsath, §,| Archil Shartava, ⊥,@ David G. Kakhniashvili, ⊥,@ and Steven R. Goodman* ,⊥,@ Mass Spectrometry and Protein Structure Laboratory, Department of Biochemistry and Molecular Biology, Department of Cell Biology and Neuroscience, and USA ComprehensiVe Sickle Cell Center, College of Medicine, UniVersity of South Alabama, Mobile, Alabama 36688 ReceiVed April 4, 2001; ReVised Manuscript ReceiVed October 24, 2001 ABSTRACT: We have previously demonstrated that an oxidative change, the formation of a disulfide bridge between two cysteine residues, in the membrane protein -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 -actin and pharmacological research toward the reversal of the oxidation, we attempted to prepare oxidized -actin from normal red blood cell (RBC) -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). Irreversibly sickled cells (ISCs) 1 of sickle cell anemic patients remain sickled even under conditions where they are well-oxygenated and hemoglobin is depolymerized (1). Earlier investigations in our laboratories have shown (2, 3) that the result of an oxidative change, a disulfide bond between 284 Cys and 373 Cys of the membrane protein -actin, distinguishes ISC -actin from normal RBC -actin. This single S-S bond not only limits the cell’s normal functions but also alters its adhesive characteristics and the fragility of the membrane, causes capillary occlusion and tissue damage, and probably is an important component of vasooc- clusive crises. It is reasonable to expect that a properly designed pharmacon could inhibit, and maybe even reverse, the oxidative reaction and ameliorate the disease (4). For experiments aimed at investigating the chemical and functional characteristics of ISC -actin, a considerable quantity of ISC -actin is needed. A polymerization experi- ment, or an association experiment (2) with spectrin, -actin, and protein 4.1, requires at least 0.1 mg of actin. To crystallize actin for X-ray crystallography, one needs several milligrams in a very pure form. We cannot expect the necessary amounts of blood from sickle cell patients, because irreversibly sickled cells make up just a small portion of the RBCs, and large volumes of blood are not available from these already anemic patients. Therefore, we attempted to generate ISC -actin from normal RBC -actin using the more readily available normal human blood as an alternative source. During the investigations, efforts were also spent to minimize the amounts of actin that were needed for the experiments. We employed reverse phase LC to purify actin for the redox reactions and chemical tests, and we imple- mented a fluorescence-based polymerization assay carried out in the microflow cell of the LC fluorescence detector. EXPERIMENTAL PROCEDURES Preparation of Normal RBC -Actin. Normal -actin was isolated from the blood of healthy adults using our previously published method (2), with a modification that replaced size exclusion chromatography with reverse phase liquid chro- matography with UV detection (LC/UV). However, in a few instances, the original size exclusion chromatography was used to obtain larger amounts of -actin. SDS-PAGE indicated homogeneous -actin. Briefly, the ghosts obtained from the RBC fraction of 20- 30 mL of blood by lysis and centrifugation were treated with a Triton X-100 buffer to remove the lipid layer. The remaining skeletons were dissociated with a Tris buffer. From the resulting mixture of skeletal proteins (spectrin, protein 4.1, and -actin), -actin was isolated by LC/UV as follows, † This work was supported by National Institutes of Health Grant 3P60 HL38639 to the USA Comprehensive Sickle Cell Center on which S.R.G. serves as Program Director. ‡ A part of this work was presented at the 48th Conference on Mass Spectrometry and Allied Topics, June 11-15, 2000, Long Beach, CA. * To whom all correspondence should be addressed. Present ad- dress: Department of Molecular and Cell Biology, The University of Texas at Dallas, Biology F03.1, Box 830688, Richardson, TX 75083- 0688. Phone: (972) 883-4872. E-mail: sgoodmn@utdallas.edu. § Mass Spectrometry and Protein Structure Laboratory. | Department of Biochemistry and Molecular Biology. ⊥ Department of Cell Biology and Neuroscience. @ USA Comprehensive Sickle Cell Center. 1 Abbreviations: ISC, irreversibly sickled cell; DTNB (Ellman’s reagent), 5,5′-dithiobis(2-nitrobenzoic acid); FAB, fast atom bombard- ment ionization; FD, fluorescence detection; LC, liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MBrB, monobromobimane; MS, mass spectrometry; RBC, red blood cell; UV, ultraviolet photometry; TCEP, tris(carboxyethyl)phosphine; TFA, trifluoroacetic acid. 292 Biochemistry 2002, 41, 292-296 10.1021/bi010685v CCC: $22.00 © 2002 American Chemical Society Published on Web 12/08/2001