Selective Ion Transport across Biological Membranes A Simple Experiment in Bioinorganic Chemistry Paul A. Adams MRC Biomembrane Research Unit, Department of Chemical Pathology. University of Cape Town Medical School. Observatory 7925, Cape Town, Republic of South Africa Duncan Black Department of General Surgery, University of Cape Town Medical School, Observatory 7925, Cape Town, Republic of South Africa occur over a period of several minutes. Microscopic examination (X 400) of the suspensionat thia point shows the erythrocytes are intact and undamaged by the lo4-or SzOs2-anions. Centrifugation at 17W g for 10 min spins down the dark-colored cells leaving a colorless supernatant. In the case of the trivalent Fe(CN)63- ion, however, addition to the red cell suspension does not change the bright red color aver a period of 2 h. Addition of one drop of detergent (Triton
Three types of transport across biological membranes can be identified. 1. Passive: where transport is a simple diffusive process across a
semipermeable membrane. 2. Active: where transport is energy-linked as in Ca2+transport by the Ca2+-ATPaseof sarcoplasmic reticulum. 3. Facilitated: where transnortis effectedutilizinea "carrier"in the cell memhrane itself. A Aeeifie examnle of this-is the transnort of mono- and divalent (hut not trivalent) anions across the cell membrane by means uf the band-3 rarrier protein, which is ". . . arguably the best understood mammalian trampun 8ystPm" ( I ) . ~
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T h e band-3 carrier protein (MW 90,00&100000 Daltons) is the most abundant protein of the red cell membrane, and convincine evidence for its involvement in anion transport has been ibtained from a variety of sources. Of p a r t i c h importance are the demonstrations that the stilbene disulfonates, which covalently bind to the carrier protein, are specific inhibitors of the erythrocyte anion exchange process (2.3). Furthermore, purified band-3 protein, when reconstituted into phospholipid vesiclen, confers an attenuated (and stilbene diaulfate inhibited) anion exchange capahility on the vesicles ( 4 , 5 ) , tho reduced exchange activity being paralleled bv a reduction in the covalent hindine of stilbene disulfonateby the protein (6). In this demonstration. which reauires n o instrumentation other than a balance, pH meter, A d bench-top centrifuge, the intact human ervthrocvte is utilized as a simple model with which to demon&rateibe essential features of facilitated anion transport across biological membranes. Prlnclple of the Demonstration This experiment is based on the bright-red to blue-purple color change associated with the conversion of oxyhemoglobin (OzFe2+Hb) to methemoglobin (Fe3+Hb)-with concomitant release of 02-mediated by a variety of mono-, di-, and trivalent oxidizing anions, e.g., periodate (104-), metabisulfite (S2052-), and ferricyanide (Fe(CNd-) (see Fig.
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Fbur~1. UV/vialble soectrum of IAl: solution wavelenoths are . . oxvhemwlobln . " i,,-~or(ltband 415 a osnd 577 nm. 3bd.a 541 om. (8):onlhemogmbln so ution (A1 r i mM 1 .0 : S205'-, or Fe(Ch).'-. A,-Sorer ban0 405 nm. visim region bands at 500 an0 631 nm Inset dilfersnce spectrum. 8 - A.
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1). Experimental Protocol 1 e m 3 heparinized fresh human1 blood is diluted to 10 em3with 0.9% (w/v) NaCl and centrifuged for 5 min at 17W g. The superuatant is discarded, and the packed red cells again washed (X 2) with 10 volumes of the saline. The washed cells are then suspended in 3 cm3 of an isotonic solution of 5 mM Hepea in saline (0.9%)buffer (pH 6.4). Transport experiments are conducted by mixing 0.5 cm3of the erythrocyte suspensionwith 0.5 cm3of the oxidizing anion solution (0.05 mol dm-3; pH adjusted to 6.5). In the case of the periodate and metabisulfitea change from hright red to blue-purple is seen to
'Possible hazards associated wlth the handling of human blood can be eliminated by having each student use his or her own blood. collected bv oricking the thumb with a sterile needle and collectina " the drops oi blood diectly into 0.9% NaCl In a centrifuge tube. 526
Journal of Chemical Education
Flgure 2. (a) Red cell suspension: (2):suspnslon (1) after 8 min exposure lo 0.025 mot d m P S2Os2-. (3):suspnsion (2) after cantrifugatlon.
XI-which dismuta cell membranes-to the red-ceU/Fe(CNIe3. ." m h m gives rhbimmediate bright.red-to-purple color change. Centrifugation at 1700 g for 30 min does not lead to any removal of color from the solution. ~~
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Conclusion Mono- and divalent oxidizing anions cross the erythrocyte cell membrane without disruption of the membrane and oxidize 02Fe2+Hbto Fe3+Hh. The intact cells can be spun down to leave a colorless solution. Trivalent oxidizing anions, of a similar size to the monoand divalent ion do not oxidize OzFe2+Hbin the intact red cell system. Upon disruption of the cell membranes and release of the oxvhemoelobin immediate conversion to Fe3+Hb is observei. On centrifugation, the soluble Fe3+Hb remains in solution. We conclude, therefore, that transport
of anions across the erythrocyte memhrane is selective, mono- and divalent anions can cross the membrane, while trivalent anions of a similar size cannot. The latter observat not oassive. tions confirmine that the t r a n s ~ o ris This experiment constitutes an extremely simple, inex~ e n s i v edemonstration of a fundamental bioinoreanic ~ r o cess and could easily be incorporated into a larger projec't, in which further characteristics and properties of blood are explored. Literature Cned 1. Jay, D.: Canthey,L. Ann. Re". Bioehem. 1986,55,511538. 2. Ha. M. K.; Guidotti, 0 . J .B i d Cham. 1375,250,676483.
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5. R0ss.A. H.; McCannell, H.M. J. B i d Chem.1978,253,4777-4782.
6. Cabanfehik,Z.I.:VoLnky,D.J.:Ginaburg,H.;h~r,A.Ann.NY.Acod.Sci.1980,341, 44M54.
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June 1990
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