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Blood, Vol. 113, Issue 24, 6237-6245, June 11, 2009
Imaging of the diffusion of single band 3 molecules on normal and mutant erythrocytes
Blood Kodippili et al.
113: 6237
Supplemental materials for: Kodippili et al
Materials used for synthesis of biotin-DIDS
Diaminodisulfonic acid stilbene (DADS) was purchased from Aldrich Chemical Company (St. Louis, MO). Amino acids and their protected derivatives (Fmoc-Lys(ivDde)-OH, Fmoc-Asp(O-2-PhiPr)-OH, Fmoc-Asp(OtBu)-OH), Fmoc-Gly-Wang resin, HBTU 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate, and HOBt (N-Hydroxybenzotriazole.H2O) were obtained from Novabiochem (Madison, WI). Other reagents were thiophosgene (FLUKA, Milwaukee, WI); DIPEA (N,N-Diisopropylethylamine) and bovine serum albumin (BSA) ( Sigma, St. Louis, MO); HATU (2-(1H-7-azabenzotriazol-1-yl)–1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium) (Applied Biosystems, Foster City, CA); streptavidin-linked quantum dots, AlexaFluor 488, AlexaFluor 598 (Invitrogen Molecular Probes); triisopropylsilane (TIS) and trifluoroacetic acid (TFA) (Aldrich, St. Louis, MO); and 1-methyl-2-pyrrolidinone (Acros Organics, New Jersey, USA). PBS (phosphate buffer saline pH 7.4) was comprised of 137 mM NaCl (sodium chloride), 2.7 mM KCl (potassium chloride), 8.1 mM Na2HPO4 (sodium phosphate), and 1.5 mM KH2PO4 (potassium dihydrogen phosphate). Synthesis of biotin-DIDS
Fmoc-Gly-Wang resin beads (50 mg) were incubated in dimethylformamide for 2h while bubbling with N2 (g) at room temperature. All reagents in the synthesis scheme (See Fig. S1) were added in the order indicated at a 2.5 molar excess to active sites on the resin and allowed to react for 2hr. Deprotection with 2% hydrazine (NH2-NH2) or 20% piperidine was done by incubating the resin with the desired reagent 3× for 5 min each. Effectiveness of deprotection was tested using ninhydrin. Coupling reaction was repeated once without further deprotection to increase yield. The product was cleaved from the resin with a mixture of 9.5:2.5:2.5 of TFA, H2O, and TIS, and purified by HPLC (High performance liquid chromatography). This penultimate compound, containing the biotin conjugated to DADS, dissolved in sodium bicarbonate (150mM) was reacted for 1h with one equivalent of aqueous thiophosgene with vigorous mixing to obtain the final product. Diethyl ether was added to the reaction mixture to extract thiophosgene and quenched it by potassium hydroxide. The biotin-DADS to biotin-DIDS conversion is done following the published protocol for DADS to DIDS (4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid) (by Mike Jennings and coworkers).The product, biotin-DIDS, was analyzed by both MALDI-TOF mass spectrometry and UV spectroscopy. The presence of the desired compound was established by observation of a prominent peak in the mass spectrum of m/z =1165.8 (calculated mass 1166) and the appearance of an absorption spectrum that mimics the composite of the UV-visible spectra for DIDS (342nm) and biotin (broad maximum between 200–250nm) (See Fig. S2).
Files in this Data Supplement:
- Figure S1. Synthesis scheme of DIDS-biotin (JPG, 420 KB)
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- Figure S2. UV spectra of DIDS derivatives used in the synthesis of the DIDS-biotin conjugate (JPG, 227 KB)
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- Figure S3. Representative images of the human RBCs analyzed (JPG, 480 KB)
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- Figure S4. Distribution of microscopic and macroscopic diffusion data on individual normal volunteers (JPG, 290 KB)
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- Figure S5. Histogram plots of band 3 diffusion data obtained on erythrocytes from hereditary elliptocytosis patient 3 (HE-3) (JPG, 157 KB)
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- Figure S6. Analysis of erythrocyte deformability as a function of osmolality by ektacytometry on HE samples 1, 2, and 3 (JPG, 345 KB)
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- Figure S7. Examination of defects in HS samples 1 and 2 by SDS-PAGE (JPG, 323 KB)
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- Figure S8. Histogram plots of band 3 diffusion data obtained on erythrocytes from HS patients 3–6 (JPG, 340 KB)
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- Figure S9. Scatter plots of compartment size vs. microscopic diffusion coefficient (JPG, 299 KB)
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