HEMATOLOGY #6 --Erythrocyte:Structure/Composition/Function

I. Erythrocyte Primary Function - Maintenance of Respiration A. Respiration: utilizes oxygen, releases CO2 , H+ 1. Transport of O2 from lung alveoli for delivery to tissue 2. Transport of CO2 from tissues for delivery to lung 3. Transport of H+ from tissues for delivery to lungs 4. 120d life ===>300 miles of travel through circulatory system B. Functions are mediated by Hemoglobin (Hb) 1. Erythrocyte is the carrier and facilitator of Hb and Carbonic anhydrase/Band 3 (Anion transport protein) which carry out the O2 and CO2 transport functions, respectively 2. Function of Hb requires no energy but function of erythrocyte does II. Reasons for compartmentalization of Hb in an Erythrocyte A. Solubility of O2 in H2O or plasma cannot support human life 1. Ambient [O2]H2O ~ 300 FM 2. Human has ~ 4-5 l of blood (4.7 l avg); therefore, O2 content of 1 volume of blood is: 4.7 (l) x 300 x 10-6 mole/l = 1.4 x 10-3 moles O2 3. 0.75 kg O2 consumed by normal adult respiration in 24h; therefore, (750g)/(32 g/mol) = 23.5 mole (O2 used)/24 h and this would require (23.5mole)/1.4 x 10-3 = 16,740 pump cycles/day 4. Entire blood supply pumped every minute; therefore, 60 cycles/hr x 24 hr = 1440 Heart would have to pump 16,740/1440 or 12 x faster. B. Hb has 4 O2 binding sites/molecule [Hb]RBC = MCH/MCV = 30 pg/90fl = 30x10-12 g/90x10-15 l = 0.33 x 103g/l MW Hb = 68,000 therefore: [Hb] = 330 g/l /68x103 g/mole = 4.9 x10-3 mole/l or ~ 5mM 1. but there are 4 binding sites/molecule of Hb 2. thus O2 capacity of the erythrocyte is = 20mM 3. since ~half the volume of blood is RBC (i.e. hematocrit of 45-50%) 4. thus the O2 capacity of the blood is 10mM or 30 - 50X that of the plasma or solubility of O2 in H2O ~ 0.2 0.3 M C. Hb must remain compartmentalized because 2.5 mM Hb in plasma would make it too viscous for efficient pumping and result in hyperviscosity syndrome and hyperosmolarity dehydrating the tissues.

D. E.

Compartmentalization within a cell requires cells to be of maximum size yet still able to get through capillaries i.e. deformable. Why the shape ? Compartmentalization allows: 1. 2. 3. 4. Sequestration of active molecules - in a functional environment Efficient recognition of inactive or damaged units Protection from toxic environment - O2 is toxic to proteins! Protection from oxygen a. O2 in the presence of Fe+2 and H2O spells trouble for proteins -- promotes generation of toxic oxygen species: OH! , O2- , 1O2 b. Haber Weiss Reaction : H2O2 + O2 - === Fe Chelate ==> O2+ OH- + OH! <==inactivates proteins c. Formation of methemoglobin (3% oxidized per day) O2 + Fe+2 C Heme ==> O2- + Fe+3C Heme<==inactive in Hb H2O2 + Fe+2 ==> OH. + Fe+3 d. Oxidation of sulfhydryls 2O2- + 2H+ ==> O2 + H2O2

<==inactivates proteins

H2O2 + R1B SH + R2BSH==> R1B SBSBR2 + 2H2O These Rx and their products will inactivate Hb, Na+K+ATPase, Band III other membrane proteins. Therefore, in the highly rich O2 environment of the RBC cytoplasm, RBC must carry protective enzymes. F. Protective Enzyme systems of the RBC-- have the highest enzyme activities 1. Superoxide dismutase -- 2,250 IU/g Hb (1 IU/1mole rx / min @37oC) 1g Hb = 15 mole + SOD 2O2 + 2H == ==> O2 + H2O2 2. Catalase -- 153,000 IU/g Hb 2 H2O2==CAT==> 2H2O + O2 3. Methemoglobin reductase -- 19 IU/ g Hb reverses the inactivation of Hb by H2O2 inactive HbBFe+3(methhemoglobin)==> active Hb-Fe+2 + H2O2 (Ferric) (Ferrous) 4. NADPH Diaphorase -- 2 IU g/Hb inactive HbBFe+3(methhemoglobin) ==>active Hb-Fe+2 + H2O2

5. Glutathione Peroxidase -- 31 IU/g Hb

H2O2 ========H2O
Glutathione peroxidase

31 IU /g Hb

GSH ========= GSSG

glutathione reductase

17 IU /g Hb

NADP =======NADPH
glucose- 6 phosphate dehydrogenase

8 IU /g Hb

Glucose 6-P ====== 6-P-Gluconate Feed from= Pentose-P === 6 phosphogluconate dehydrogenase === 6-P-Gluconate Hb anaerobic glycolysis HEXOSE MONOPHOSPHATE SHUNT (no mitochondria) 6. Glutathione -- high protective concentration of 2 mM t1/2 4 days a. removes H2O2 2GSH + H2O2 ==> GSSG + 2H2O b. protects protein (Hb) sulfhydryls 2GSH + R1BSSBR2 ==> R1BSH +R2BSH +GSSG c. GSSG is transported out of RBC or converted to GSH by HMPS (above) in RBC d. Structure ECG glutamate-cysteine-glycine 9 IU /g

G.

Main RBC energy requirement is: HMP Shunt Meth Hb Reductase GSH resynthesis Glutamate + cysteine + ATP ==> glu-cys + ADP+P1 Glutamyl Cysteine + glycine + ATP ==> GSH + ADP + P1 GSSG ATPase GSSG transport out of RBC 3Na+ out 2K+ in = Isoosmotic - Hb content Na+K+ATPase 5X osmotic pressure of plasma Intracellular [Na+]=15mM ; [K+]=80mM Extracellular [Na+]=150mM ; [K+]=5mM Ca++ATPase Ca++ out==>leakage in===>echinocytes ++ Mg ATPase

H. J.

All energy provided by anaerobic glycolysis Interesting numbers CONCENTRATION % OF TOTAL % OF TOTAL PROTEIN -------------100 % 97.5 % 1.6 % 0.8 %

RBC CONSTITUENT H2O Protein Hb Inert protein Enzymatic protein

721 mg/ml 371 mg/ml 362 mg/ml 6.3 mg/ml 2.9 mg/ml

66 % 34 % 33 % 0.5 % 0.3 %

III. The Compartmentalization System A. The RBC Membrane - The most studied cell membrane 1. 2. 3. 4. Membrane Barrier separation plasma & high Hb inside RBC Maintains intra corpuscular environment Holds - pumps and channels for Na+, K+, Ca++, G-SS-G, glucose, anions Maintains flexible shape

B. RBC Membrane Composition 1. Lipids -- 50% Membrane weight is lipid -- 95% phosopholipids & cholesterol a Cholesterol phospholipid ratio ~ 1:1 b. Major phospholipids -phosphatidyl choline (on outer leaflet)-- 30% -phosphatidyl ethanolamine (inner) --28% -phosphatidyl serine (inner)-- 14% -sphingomyelin --25% (nonglycerol derived) -lysolecithin (collect at phase interfaces) -- 2%

2. RBC Membrane Proteins -- 50% Membrane weight is protein a. SDS-PAGE analysis of erythrocyte membranes b. 10 major, 200 minor bands c. peripheral membrane proteins are bound to the inner or outer leaflet by ionic or receptor-ligand-like interactions d. integral membrane proteins have highly hydrophobic stretches that span the membrane and are therefore transmembrane proteins; or linked by lipid anchors; e. often glycosylated on extracellular face

Band #

Name (MW)

Type

# Copies/C ell 2 x 105

Functions

Band 1 Band 2 Band 2.1, 2.2, 2.3, 2.6 Band 3 PAS-1

Spectrin (240K) (215K) Ankyrin (195K) Anion Exch. Protein Glycophorins A, C

inner extrinsic inner extrinsic transmmbrane transmmbrne inner extrinsic

x-linking protein for MSK x-links spectrin to Band 3 Membrane anchor for MSK, anion exc Membrane anchor for MSK links to Band 4.1

1 x 105

1.2 x 106

4.1

(80K)

2 x 105

x-links actin trophomyosinspectrin complexes with glycophorin C x-links actin trophomyosinspectrin complexes with glycophorin C integral membrane glycoprotein

4.2

(72K)

transmmbrne

2 x 105

PAS-2 Band 5 Band 6

Glycophorin A monomer (43-48 K) Actin (43K) Glyceraldehyde-3 phosphate dehydrogenase Globin of (Hb) (18K)

transmmbrne inner extrinsic inner extrinsic inner extrinsic 5 x 105

Complexes with tropomyosin 4.1

Complexes with membrane

3. Membrane Organization a. bilayer with laterally mobile lipids and transmembrane proteins b. asymmetric PC outside, PE, PS inside, cholesterol equal c. inner surface membrane skeleton - insoluble in mild detergents - self associating oligomers of spectrin link to membrane integral membrane proteins by linking proteins c. formation of discrete lateral lipid and protein domains and an elegant chainlink-like network called the membrane skeleton

4. Functions a. carries unesterified cholesterol b. carries lysophospholipids c. impermeant barrier to to polar molecules (water soluble) d. w/ MSK provides integrity, flexibility, deformability, integrity e w/ transport protein contols RBC volume, Ca++ homeostasis f. carries out anion exhange allow Cl-/HCO3- exchange and CO2 ransport