Диелектрични релаксации в еритроцитната мембрана проявяващи се при денатурацията на спектрина

1. Диелектрични релаксации в еритроцитната мембрана проявяващи се при денатурацията на спектрина Иван Танев Иванов, Бояна Първанова I.T. Ivanov, B. Paarvanova, Dielectric relaxations on erythrocyte membrane as revealed by spectrin denaturation, Bioelectrochemistry, Volume 110, August 2016, Pages 59–68, (Impact factor 2015/2016 - 3.556)

2. 1 – α-spectrin monomer 2 – β-spectrin monomer 3 - polymerized actin 4 - band 3 dimer 5 - band 3tetramer 6 – ankyrin 7 - glycophorin C 8 - band 4.1 9 - lipid bilayer Fig. 1.A schematic diagram of human erythrocyte membrane (Tse and Lux, 1999).

3. МАТЕРИАЛИ И МЕТОДИ • Получаване на еритроцити, изолирани еритроцитни мембрании изолирани подмембранни цитоскелети (Тритонови скелети) • Прекъсване на връзката спектрин-анкирин-анионен обменник • При алкално рН на средата (рН 9.2) • Обработване на еритроцитите с DIDS • Инкорпориране на 2,3-DPG в изолираните и затворени мембрани • Прекъсване на връзкатаспектрин-актин • Инкорпориране на DNAse I в изолираните и затворени мембрани • Термични и диелектроскопични методи за анализ

4. The resealedmembranes contained 150 mM NaCl and were suspended in isotonic solution of60 mM NaCl and sucrose, pH 7.0. Hematocrit and heating rate were 0.07 and2.0°C/min. FIG. 2. DERIVATIVE THERMOGRAM OF THE ELECTRIC CONDUCTIVITY OF SUSPENSION,CONTAINING ISOLATED RESEALED MEMBRANES (EMs) OF HUMAN ERYTHROCYTES

5. The suspension contained human erythrocytrs (left) and EMs (right). The suspension media contained various concentrations of NaCl and sucrose, in mOsm FIG. 3. EFFECT OF THE TRANSMEMBRANE GRADIENT OF ION CONCENTRATION ON THE DERIVATIVE THERMOGRAM OF SUSPENSION CONDUCTIVITY

6. Fig. 4. SPECIES VARIATIONS OF ERYTHROCYTE MEMBRANE THERMOSTABILITY, Tgo, (A panel) AND LIFE SPAN OF ERYTHROCYTES IN THE CIRCULATION OF MAMMALS (B panel) (A): The concentrations of phosphatidyl choline (PC) and sphingomyelin (Sph) are in % oftotal membrane phospholipids. (B): Mature erythrocytes live on average 153 days in sheep; 110 days in dog; 140 days in cow and 100 days in horse;120 days in human; 65 days in pig and 125 days in goat (Tsolov et al.,1985); 8 weeks in rabbit; 54 days in rat, 36 days in mouse and 30 days in guinea pig.

7. ●— 50 kHz ○— 100 kHz ■— 400 kHz □— 800 kHz ▲— 1400 kHz Δ— 2000 kHz ♦— 3000 kHz ◊— 8000 kHz Fig. 5. Effect of frequency on the temperature profile of real impedance, Zre, of EMsuspension. The arrows indicate the spectrin denaturation temperature, TA andthe temperature, TG, of threshold hemolysis.

8. Shown is the−ΔZim vs. ΔZre dependence, where ΔZim (Ω) and ΔZre (Ω) are the corrected changes incomplex suspension impedance, associated with the denaturation of spectrin. Thesuspension contained EMs (open circles) and Triton-X-100 residues of EMs (blackcircles). FIG. 6. COMPLEX IMPEDANCE (NYQUIST) PLOT AT THE TEMPERATURE OF SPECTRIN DENATURATION

9. Thesuspension contained: • Intact erythrocytes (open circles) • DIDS-inhibited erythrocytes with disconnected band3-ankyrin bridges (gray circles) • DNAase-incorporated EMs with depolymerized actin (black circles) FIG. 7. COMPLEX IMPEDANCE (NYQUIST) PLOT AT THE TEMPERATURE OF SPECTRIN DENATURATION

10. The critical frequency, fgamma (MHz), isplotted against the NaCl concentration, [NaCl] (mM). The EMs (●) contained theindicated concentration of NaCl. The Triton-X-100 shells (■) were dispersed ina solutionwith the indicated NaClconcentration. FIG. 8. EFFECT OF NaCl CONCENTRATION ON THE CRITICAL FREQUENCY, FGAMMA, OF RESEALED EMs (■)AND OF THEIR TRITON-X-100 INSOLUBLE RESIDUES (●)

11. The energyloss is expressed by the imaginary impedance, Cim (pF), plotted against the frequency ofelectric field, f (MHz). • The temperature, hematocrit and suspension medium were 20°C,0.60 and a solution of 5 mM NaCl and 140 mM mannitol, respectively. • The straight linecorresponds to the conduction loss and shadowed area represents the dielectric loss dueto dipole relaxation. FIG. 9. EFFECT OF FREQUENCY ON THE ELECTRIC ENERGY DISSIPATED BY DIPOLES OF SUSPENED EMs

12. Thedielectric loss, Cim–Cim(ionic) (pF)is plotted against the frequency, f (MHz). Fig. 10. Effect of temperature on the frequency profile of dielectric loss on EMs Curve d is obtained subtracting the curve bfrom the curve a′.

13. Solartron SI 1260 честотен анализатор на импеданса (Schlumberger Instruments, Хампшир, Англия)

14. БЛАГОДАРЯ ЗА ВНИМАНИЕТО