Spectrin isolation and biotinylation

Erythrocyte ghosts were obtained from whole human blood by rupturing the erythrocytes by hypotonic lysis (Dodge et al., 1963). Twelve 6ml ACD tubes (BD Vacutainer Systems, Plymouth, UK) containing human blood donated by volunteers, were centrifuged at 800 g for 10 minutes at 4 ºC using a Jouan BR 3.11 centrifuge (Jouan Inc., Winchester, USA). The plasma and buffy coat were removed and the erythrocytes washed three times in cold 0.9 % sodium chloride.

Erythrocytes were lysed with 30 volumes of cold erythrocyte lysis buffer (3 mM sodium phosphate buffer, pH 8, 0.1 mM Na2EDTA, 0.1 mM phenylmethanesulphonyl fluoride (PMSF; see Appendix A1 for stock solution;

Roche Diagnostics Gmbh, Mannheim, Germany) and centrifuged at 25,000 g for 15 minutes at 4 ºC using a Beckman^® J2-21 centrifuge (Beckman Coulter, Inc., Fullerton, USA). The supernatant and the residual white cell pellet were removed and the ghosts washed a further three times with erythrocyte lysis buffer. Spectrin

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was extracted from the erythrocyte ghosts by low ionic strength extraction (Coetzer and Palek, 1986). This was achieved by adding 30 volumes of fresh spectrin extraction buffer (0.1 mM sodium phosphate, pH 8, 0.1 mM Na2EDTA, and 0.1 mM 1,4-dithiothreitol (DTT; Roche Diagnostics Gmbh, Mannheim, Germany) and centrifuging at 30,000 g for 30 minutes at 4 ºC. DTT prevents disulphide bond formation and thereby inhibits the aggregation of spectrin. The supernatant was aspirated until there was a 1:½ ratio of erythrocyte ghosts to buffer remaining in the tube. The suspension was incubated overnight at 4 ºC.

Pefabloc SC (an irreversible protease inhibitor) (Roche Diagnostics Gmbh, Mannheim, Germany) was added to a concentration of 0.1 mM. The sample was centrifuged at 150,000 g for 30 minutes at 4 ºC, using a Beckman^® L8-70M ultracentrifuge (Beckman Coulter, Inc., Fullerton, USA) and the supernatant containing the crude spectrin extract collected and pooled.

Size exclusion chromatography and concentration of spectrin

Spectrin tetramers and dimers were removed from high molecular weight complexes and band 4.1 by size exclusion chromatography (Zail and Coetzer, 1984). The crude spectrin sample was incubated at 37 ºC for 30 minutes to dimerise the spectrin and loaded onto a 3 cm x 100 cm Sepharose^® (CL) 4B column (kept at 4 ºC) (Pharmacia Fine Chemicals AB, Uppsala, Sweden) equilibrated with spectrin column buffer (10 mM sodium phosphate buffer, pH 7.6, 130 mM KCl, 20 mM NaCl, 1 mM Na2EDTA, 1 mM DTT) at a flow rate of 20 ml/hr. The elution profile of the collected fractions (2.5ml per tube) was plotted using a LKB Bromma 2238 Uvicord S II UV reader (Pharmacia Fine Chemicals AB, Uppsala, Sweden) (settings: 280 nm, absorbance 0.5) and a LKB Bromma 2210 Recorder (settings: 0.5mm/min, 500mV). Fractions containing only the spectrin dimers were pooled and the concentration of a 10 µl sample determined with the Coomassie Plus™ Protein Assay Reagent (Pierce Biotechnology Incorporated, Rockford, USA). Spectrin tetramers were not used for the biotinylation procedure because they eluted just after the high molecular weight complexes and contamination with other erythrocyte membrane proteins 46

was possible. The spectrin dimers were concentrated to 2.5 ml using an Amicon Stirred Ultrafiltration Cell kept on ice (Model 8050; Millipore Corporation, Bedford, USA) that contained an Ultracel Amicon YM 10 Disc Membrane (molecular weight exclusion limit of 100,000 Da; Millipore Corporation, Bedford, USA). The concentration procedure was performed with nitrogen (African Oxygen Limited, Johannesburg, South Africa) set at 0.7 atm. The sample was further concentrated to ~5 mg/ml with a 0.5-3 ml Slide-A-Lyzer^® Dialysis cassette (Pierce Biotechnology Incorporated, Rockford, USA) and Polyethylene glycol 2000 (PEG 2000) (Sigma-Aldrich Corporation, St. Louis, USA).

Biotinylation of purified spectrin

The concentrated spectrin sample was dialysed against phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.2) for 2-3 hours at 4 ºC using a 0.5-3 ml Slide-A-Lyzer^® Dialysis cassette. The spectrin was biotinylated for 2-4 hrs at room temperature with continuous stirring in a reaction that contained a molar ratio of 1:10 of D-biotin-N-hydroxysuccinimide ester (Roche Diagnostics Gmbh, Mannheim, Germany) to spectrin dimers (MW 460 kDa) (Figure 19).

Figure 19: The biotinylation reaction.

(adapted from Roche Applied Science, 1999)

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The sample was dialysed overnight at 4 ºC against PBS to remove free biotin and centrifuged at 8,500 g for 10 minutes at room temperature using a Sorvall^® RMC-14 Refrigerated Microcentrifuge (Sorvall Products, L.P., Newtown, USA). The supernatant was diluted to a final concentration of 1 mg/ml with PBS. One hundred microlitre aliquots were stored at -20 ºC.

Analysis of spectrin

The biotinylated spectrin was electrophoresed on a Laemmli SDS-polyacrylamide gel (Laemmli, 1970) and a gradient Fairbanks SDS-polyacrylamide gel (Coetzer et al., 1988, Fairbanks et al., 1971) to analyse its purity.

Ten micrograms spectrin and 45 µg erythrocyte membrane proteins were prepared by adding 5 x Suspension Solution (50 mM Tris-HCl, 5 mM Na2EDTA, 5 % SDS (w/v), 25 % sucrose (w/v), pH 8), 40 x sucrose/dye (2.5 % sucrose (w/v), 0.5 % bromophenol blue (w/v)) and 2 % β-mercaptoethanol (v/v) and heating the sample for 2 minutes in boiling water. A 14 cm x 16 cm x 1.5 mm Laemmli SDS-polyacrylamide gel containing a stacking gel composed of 4 % SDS-polyacrylamide (29.2 % acrylamide/0.8 % bisacrylamide (w/v) stock solution) in lower gel buffer (375 mM Tris-HCl, pH 8.8) and 0.1 % SDS (w/v) and a resolving gel composed of 12 % polyacrylamide in upper gel buffer (125 mM Tris-HCl, pH 6.8) and 0.1 % SDS (w/v), was cast and the spectrin electrophoresed against the erythrocyte membrane proteins at 75 V for 17 hours with Laemmli running buffer (25 mM Tris, 192 mM glycine, 0.1 % SDS (w/v). The protein bands were stained with Coomassie Blue stain (0.05 % Coomassie Blue R-250 (w/v) (BDH Laboratory Supplies, Poole, UK), 25 % isopropanol (v/v), 10 % glacial acetic acid (v/v)).

A 14 cm x 16 cm x 1.5 mm Fairbanks SDS-polyacrylamide gel was set up with an exponential polyacrylamide gradient of 3.5-17.5 % (40 % acrylamide/1.5 % bisacrylamide (w/v) stock solution) in Tris acetate buffer (40 mM Tris, 20 mM sodium acetate, 1 mM Na2EDTA, pH 7.4) and 0.2 % SDS (w/v). Protein samples were solubilised the same way as for the Laemmli SDS-polyacrylamide gel. Five 48

micrograms spectrin was electrophoresed against 20 µg erythrocyte membrane proteins at 45 V for 17 hours using Tris acetate buffer containing 0.1 % SDS (w/v). The protein bands were stained with Coomassie Blue stain.

To test successful biotinylation of spectrin, one well from a Streptawell plate (transparent, 12x8-well strips; Roche Diagnostics Gmbh, Mannheim, Germany) was coated with 0.5 µg of biotinylated spectrin in 200 µl Tris buffered saline (TBS; 10 mM Tris-HCl, 150 mM NaCl, pH 8), by allowing the protein to bind for 1 hour at 4 ºC. A second control well was incubated with 200 µl TBS. The wells were washed 3 times with 300 µl TBS-T (TBS with 0.12 % Tween^®-20 (v/v) (Calbiochem^®, San Diego, USA), blocked with 200 µl 3 % Bovine Serum Albumin Fraction V (w/v) (Roche Diagnostics Gmbh, Mannheim, Germany) in TBS and analysed with a 1:100 dilution of a rabbit α-spectrin and rabbit anti-β-spectrin primary polyclonal antibody mix (prepared at St. Elizabeth’s Medical Center, Boston, USA), followed by a 1:1000 dilution of goat anti-rabbit IgG peroxidase (Roche Diagnostics Gmbh, Mannheim, Germany). The secondary antibody was detected with 0.5 mg/ml 4-chloro-1-napthol (Sigma-Aldrich Corporation, St. Louis, USA) and 0.1 % hydrogen peroxide solution (Sigma-Aldrich Corporation, St. Louis, USA).