Professional Documents
Culture Documents
Chapter 10 - The Mature Erythrocyte
Chapter 10 - The Mature Erythrocyte
Chapter 10 - The Mature Erythrocyte
Structural Features
The mature human erythrocyte is one of the most highly specialized of
cells, consisting of a plasma membrane surrounding a solution of
protein and electrolytes. More than 95% of the cytoplasmic protein is
the oxygen-transport protein, hemoglobin. The remainder includes
those enzymes required for energy production and for the maintenance
of hemoglobin in a functional, reduced state. Lacking such cytoplasmic
organelles as nucleus, mitochondria, or ribo-somes, the red cell is
unable to synthesize new protein, carry out the oxidative reactions
associated with mitochondria, or undergo mitosis. Nevertheless, the
red cell subserves a number of crucial and complex functions in the
human body.
Shape
At rest, the normal human erythrocyte is shaped like a flattened,
bilaterally indented sphere, a shape often referred to as a biconcave
disk (Fig. 10.1) . In fixed, stained blood smears, only the flattened
surfaces are observed; hence, the erythrocyte appears circular, with a
diameter of about 7 to 8 mum and an area of central pallor
corresponding to the indented regions, on fixed blood films.
The disk shape is well suited to erythrocyte function. The ratio of
surface to volume approaches the maximum possible value in such a
shape , thereby facilitating gas transfer. Furthermore, the biconcave
[1]
disk is more deformable than a sphere and can undergo the changes in
shape necessary for optimal movement within the microvasculature .
When red cell movements within small blood vessels were observed by
cinemamicrography , the plane of the biconcave disk was found to be
oriented in the direction of flow. The leading edge became pointed and
the following edge blunted (Fig. 10.2) ; thus, the shape was similar to
that of a parachute or torpedo viewed from the side. When deformed in
this way, the erythrocyte can pass through a vessel of about 4 mum in
maximum diameter.
Hypotheses have been advanced to explain how the cell
[2]
[3]
194
Figure 10-1 The normal mature erythrocyte as visualized by the scanning electron
microscope ( 9800). (Courtesy of Dr. Wallace N. Jensen.)
produces and maintains the resting biconcave shape [13] [20] . The possible forces that interact to produce this
shape include ( a) elastic forces within the membrane, ( b) surface tension, ( c) electrical forces on the
membrane surface, and ( d) osmotic or hydrostatic pressures [4] . The observation that the dimple region can
easily change its location from one site to another on the membrane [5] excludes explanations of red cell
shape based on a nonuniform membrane structure [6] [7] [8] [9] .
The normal biconcave discoid red cell possesses 40% more membrane
than is needed to enclose its cytoplasmic contents in a sphere .
Thus, the shape the erythroid membrane assumes probably meets the
requirement that this excess membrane is distributed in a manner
requiring as little energy as possible. Thus far, the factors acting within
the membrane to maintain this shape, as well as those that act on it,
are still only partially understood. Early studies of the membrane
properties that would lead to assumption of a biconcave shape
demonstrated that if a sphere with a thin-walled shell of appropriate
composition were to lose volume, a biconcave shape would be the
predicted result . The properties of such a shell would be relatively
greater resistance to bending forces and relatively little resistance to
shear forces, as the biconcave shape absorbs the least bending energy.
For a membrane to exhibit these characteristics, it must be anisotropic,
that is, its physical characteristics are not the same in all three
dimensions.
Resistance to bending without shear resistance of the erythrocyte
membrane is attributable at least in part to its phospholipid bilayer
; however, simple phospholipid vesicles do not become biconcave
when deflated. The cytoskeleton, formed by a gel-like network of
proteins, undoubtedly contributes to the bending energy necessary for
assumption of the biconcave shape, as well as to membrane stability
. Abnormalities in cytoskeletal proteins cause a variety of
pathologically shaped red cells, including spherocytes and elliptocytes
. In addition, proteins adsorbed to the outer surface of the red cell,
especially albumin, may also play a role in both maintaining normal
[10]
[11]
[11] [12]
[13]
[14]
cell shape and effecting changes in that shape under some conditions.
Thus, red cells suspended in isotonic medium tend toward an
echinocytic shape until albumin is added, and increasing amounts of
albumin move cells toward the discoid shape .
The erythrocyte is remarkable for its ability to maintain membrane
integrity while exhibiting extreme deformability under normal
physiologic circumstances . Without undergoing extensive
remodeling, the erythrocyte membrane withstands high shear stresses,
rapid elongation and folding in the microcirculation, and deformation
as the erythrocyte passes through the small fenestrations of the
spleen. Cell deformability depends on both the membrane and the
cytoplasm; however, the cytoplasm of normal erythrocytes (as
opposed to sickled erythrocytes, for example) acts as an ideal liquid
and, at physiologic concentration, has very low viscosity . Thus, it is
the elasticity and viscosity of the membrane that are crucial for
deformability.
At least three sets of circumstances result in a spherical erythrocyte
shape: osmotic (hypotonic) swelling, discocyte-echinocyte
transformation, and discocyte-stomatocyte transformation. To a point,
all three types of shape change are reversible.
Osmotic swelling occurs when erythrocytes are suspended in
hypotonic solutions. Under such circumstances, the cell acquires water
and swells, first becoming cup-shaped and then spherical. These
changes are associated with an increase in volume while the cell
surface area remains the same or increases only slightly .
[15]
[16]
[16]
[17]
[18]
Figure 10-2 Human erythrocytes flowing through a vessel about 7 mum in diameter.
Direction of flow is indicated by the arrow. (By permission from Skalak R, Branemark
P-I. Deformation of red blood cells in capillaries. Science 164:717, 1969.)
195
[20]
[21]
[17]
[20]
[23]
Dimensions
The dimensions of the red cell in the living state can be estimated by
measurements made in photomicrographs (Fig. 10.4)
. For this
purpose, the cells are suspended in isotonic solutions and
photographed on edge. With ordinary light microscopy, the potential
error associated with imprecision of focus is about 0.5 mum. This value
can be reduced to 0.02 mum by means of holography with
[17] [18] [24]
With drying and staining, the cell shrinks so that diameters measured
in fixed preparations are somewhat smaller than those of cells in
isotonic solutions. The painstaking studies of Price-Jones yielded an
average normal value for red cell diameter of 7.2 to 7.4 mum .
Indirect measurements of cell volume (see Chapter 2) are consistent
with values obtained using the microscopic method. Average values for
the mean cellular volume in normal subjects range from 85 to 91 fl,
depending on the combination of methods used (Appendix A). The
variation in cell size can be documented by means of a frequency
distribution curve of red cell volumes generated from the output of a
[25]
[27]
[26]
Figure 10-5 Frequency distribution curve of erythrocyte volume. The cells are
normally distributed about a mean volume of 90 fl. (Modified with permission from
Bessman JD, Johnson RK. Erythrocyte volume distribution in normal and abnormal
subjects. Blood 46:369, 1975.)
196
the volume of the red cell column observed in a hematocrit tube is occupied by erythrocytes [28] .
Total hemoglobin content and red cell volume vary considerably more
than does hemoglobin concentration . It has been proposed that
mature red cell size and hemoglobin content are primarily dependent
on erythroid precursor cell size at the last cell division during
erythropoiesis . Reticulocytes are 24 to 35% larger than mature red
cells, although they have similar total hemoglobin content (and thus a
lower hemoglobin concentration) .
[29]
[29]
[30]
[32]
[33] [34]
[35]
[37]
[38]
Ultrastructure
In thin sections of erythrocyte membrane, fixed with either osmium
tetroxide or potassium permanganate, three distinct layers are
observed. Two electron-dense (osmophilic) layers approximately 2.5
nm (25 A) in thickness are separated by an electron-penetrable layer
about 2.0 nm thick, for a total thickness of some 7.0 nm . This
appearance has often been cited in support of the lipid bilayer
structure, with the electron-dense areas representing either membrane
protein layers or the polar ends of the phospholipids .
With air-dried, metal-shadowed red-cell membranes, features of the
surface are made apparent . In such preparations, plaques about 3.0
nm thick and 10 to 50 nm in diameter are randomly distributed over
the surface. These observations have been used to suggest the
existence of lipid-protein subunits.
Still another technique used in electron microscopic analysis of
membranes is that of "freeze-cleaving" . Erythrocytes are frozen
rapidly at -150 C and fractured with a razor blade. The cleavage plane
follows pathways of least resistance, often exposing large areas of
membrane. These surfaces are replicated with condensed carbon and
platinum, and the replicas are examined with the electron microscope.
Two types of membrane surfaces are observed with this technique.
Both surfaces are characterized by the presence of particles
approximately 10 nm in diameter. The two surfaces differ in that one
has four to five times as many of these particles as the other (2600 to
3800/mum2 as compared with 575 to 1400/mum2 ). Membrane
cleavage likely occurs in the nonpolar region between the two lipid
layers, the particles representing proteins suspended in the lipid layer,
as pre dicted by the fluid mosaic model.
The "quick-freezing" and "deep-etching" method of examining unfixed
erythrocytes by electron microscopy has succeeded in providing a
three-dimensional picture of the membrane cytoskeleton, whose
proteins are organized into structured but deformable arrays .
[39]
[40]
[41]
[42]
[43]
[44]
[44] [45]
Lipid Composition
Virtually all of the lipids in the mature erythrocyte are found in the
membrane . Qualitative and quantitative analyses have been
performed, and the data have been the subject of several reviews
. These results are summarized in Table 10.1 . Values in children
differ only slightly from those found in adults .
The majority of erythrocyte membrane lipids are either phospholipids
or unesterified cholesterol, which are present in approximately
equimolar quantities. Four classes of compounds account for most of
the phospholipid: phosphatidylcholine (lecithin),
phosphatidylethanolamine, sphingomyelin, and phosphatidylserine
(Table 10.1) . Two fatty-acid side chains are attached to all of these
lipids except sphingomyelin, which has only one. In addition, trace
amounts of other phospholipids containing only one fatty acid
("lysophospholipids," e.g., lysolecithin) or having a vinyl ether
(plasmalogens) in place of a fatty acid are found.
Phospholipids are distributed asymmetrically between the two lipid
layers of the membrane
. Eighty percent
[44]
[46] [47]
[51]
[52] [53]
197
(mumol/1010
cells)
% of Total
Weight Concentration47
(mg/1010 cells)
Phospholipids
Phosphatidylcholine (lecithin)
1.3
1.0
Phosphatidylethanolamine (cephalin)
1.2
0.9
Sphingomyelin
1.0
0.8
Phosphatidylserine
0.6
0.4
Lysolecithin
0.04
--
Others
0.07
--
% of Total
Weight Concentration47
(mumol/1010
cells)
% of Total
Total phospholipids
4.2
49.5
3.1(1.7-3.2) *
69
Cholesterol
4.0
47.1
1.3(1.1-1.4) *
29
Glycolipids (globoside)
0.21
3.4
0.1
Total lipids
8.41
100
4.5(3.9-5.2) *
Lipid
(mg/1010 cells)
% of Total
2
100
*Range in parentheses.
or more of the aminophosphatides (phosphatidylethanolamine and phosphatidylserine) lie within the inner
(cytoplasmic) monolayer, whereas the choline-containing lipids (phosphatidylcholine and sphingomyelin)
are the major components of the outer monolayer (Fig. 10.6) . Phosphatidylserine is not detectable at all in
the outer lipid layer. The functional importance of the asymmetric distribution of phospholipids in the two
erythrocyte membrane lipid layers is not completely understood, although study of certain pathologic
conditions has provided clues to both the role and regulation of this asymmetry. For example, when
erythrocytes containing hemoglobin S and little or no hemoglobin A are subjected to reduced oxygen
tension, the outer lipid layer shows increased amounts of phosphatidylethanolamine and
phosphatidylserine, whereas the distribution of sphingomyelin is unchanged [48] . The change in lipid
sidedness correlates with loss of the ability to undergo echinocyte transformation, implicating a role for
lipid distribution in regulating shape and also implying that the regulation of distribution of some
phospholipids may be independent of the regulation of distribution of others.
The lateral mobility of lipids in the outer membrane layer exceeds that
of lipids in the inner layer. Although cholesterol is known to restrict lipid
lateral mobility , the outer lipid layer likely is relatively enriched in
cholesterol . Some investigators think that lipids in the inner layer
are restricted in their mobility because of interactions of phospholipids
with cytoskeletal proteins
.
An additional effect on lipid mobility and membrane deformability may
come from the fact that the fatty acids found in erythrocyte
phospholipids also are not distributed evenly between the two bilayers
. Overall, about one half of the fatty acids in the membrane
are unsaturated (Table 10.2) . Unsaturated fatty acids, however, and
particularly the polyunsaturated acyl chains with four or more double
bonds, are a disproportionately large part of the inner leaflet
phospholipids, phosphatidylethanolamine and phosphatidylserine. In
contrast, phosphatidylcholine, which is predominantly in the outer lipid
layer, contains most of the shorter-chain saturated fatty acids.
Sphingomyelin is especially
[54]
[55]
[56] [57]
Figure 10-6 Distribution of erythrocyte phospholipids between the inner and outer
layers of the membrane. TPL, total phospholipid; SM, sphingomyelin; PC,
phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine. (From
Rothman JE, Lenard J. Membrane asymmetry. Science 195:743, 1977, with
198
Molecular Designation *
Percent
Palmitic
16:0
24.5
Stearic
18:0
19.0
Saturated
Others
3.1
Total saturated
46.6
Unsaturated
Oleic
18:1
16.4
Linoleic
18:2
11.2
Arachidonic
20:4
15.1
Others
10.3
Total unsaturated
53.0
*The first number indicates the number of carbons; the second, the number of double bonds. (From Ways P,
Hanahan DJ: Characterization and quantification of red cell lipids in normal man. J Lipid Res 5:318,
1964, with permission.)
enriched in fatty acids with a chain length longer than 20. Membranes rich in sphingomyelin are less "fluid"
than those with relatively larger amounts of lecithin [58] . An increased ratio of sphingomyelin to lecithin is
found in abetalipoproteinemia and probably accounts for the erythrocyte abnormalities associated with that
disorder [58] .
[56]
[5] [62]
[63]
[45]
[60]
LPC + PE; 7, PE
LPE +
FFA; 7b, PC
LPC + FFA. (By permission from Shohet SB. Hemolysis and changes in
erythrocyte membrane lipids. N Engl J Med 286:577, 1972.)
199
and 3). The rates of transfer are functions of the relative plasma and red cell levels of these lipids and are
indirectly affected by the activity of the cholesterol esterifying enzyme in plasma, lecithin-cholesterol
acyltransferase (LCAT) [64] . This enzyme catalyzes the reaction in which a fatty acid in the 2 position of
lecithin is transferred to free cholesterol, forming cholesterol ester and lysolecithin (Fig. 10.7 (Figure Not
Available) , reaction 1A). Neither of the LCAT reaction products can enter the membrane. In patients with
congenital LCAT deficiency, membrane cholesterol and lecithin are increased and the red cells are target
shaped [65] .
The exchange of cholesterol and lecithin between red cells and plasma
is also affected by the plasma bile salt concentration . If erythrocytes
are incubated in normal plasma to which bile salts have been added,
the cells acquire cholesterol, and this change is accompanied by an
increase in surface area and the formation of target cells. Although the
mechanism of bile salt action is not fully understood, at least two
properties appear important: bile salts inhibit the LCAT reaction and, in
addition, they bring about a shift in the distribution of free cholesterol
between plasma and cell.
Phospholipids also may be added to the membrane by three other
reactions. Albumin-bound lysophospholipid may be transferred to the
membrane (Fig. 10.7 (Figure Not Available) , pathway 4) and acylated
(reactions 5a and 6a) to form a complete phospholipid . Of lesser
quantitative importance is the transfer and conjugation of two
lysophospholipid molecules to yield a phospholipid (reaction 5b) and
glycerylphosphorylcholine (GPC), which returns to the plasma .
Finally, some phosphatidylethanolamine (PE) is produced by
transacylation of a fatty acid from lecithin to lyso-PE (reaction 6b). A
congenital defect in the last reaction results in the formation of cells
that possess increased membrane lecithin and decreased membrane
PE . These changes are associated with the clinical picture of
nonspherocytic hemolytic anemia.
The fatty acid composition, but not the major relative proportions of
the phospholipid classes, may be altered by diet . With low-fat diets,
linoleic acid levels decrease. With diets high in linoleic acid, the
amount of red cell linoleic acid increases. These changes occur
relatively slowly, over about 4 to 6 weeks.
Relatively little is known about the pathways of renewal of glycolipids.
They appear to be synthesized in the bone marrow and incorporated
into the membrane before release of the mature red cell . It is
possible that they remain with the cell throughout its life span. The
enzymatic degradation of glycolipids occurs in macrophages by
sequential cleavage of the hexose units. These reactions, and diseases
that affect them, are discussed later in this book.
[66]
[60]
[67]
[68]
[69]
[45]
Membrane Proteins
Early in the history of membrane biochemistry, erythrocytes became
the model system for the study of plasma membranes because they
lacked organelle and nuclear membranes. Solubilization of membrane
200
Molecular Weight
Percent of
Protein
Name
240,000
15}
225,000
15
beta
2.1
206,000
}5
Ankyrin
Peripheral
2.2
190,000
90-105,000
24
Anion Channel
Integral
4.1
78,000
4.2
4.2
72,000
5.0
Protein kinase
4.5
45-75,000
5.0
Glucose transporter
Integral
43,000
4.5
Actin
Peripheral
35,000
5.5
G-3PD
Peripheral
29,000
3.4
39,000
6.7
Glycophorin A
Integral
PAS-1}
Spectrin{alpha
Relation to
Membrane
Peripheral
PAS-2
Coomassie technique [71] . Originally, the seven major protein bands were referred to by their numeric
designation (Table 10.3) As further refinements in SDS-PAGE have produced greater resolution, other
bands have been given decimal or alphanumeric designations, such as bands 2.1 and 4.2, sometimes with
even further divisions such as in 4.1a and 4.1b. At present, many of these proteins are no longer identified
by this numeric nomenclature because they have been given specific names as their chemical structures
have been defined and, in many cases, their cDNAs and genes have been cloned.
[35]
Furthmayr73,75
Dahr et al.74
Anstee et al.72
PAS-1
Glycophorin A homodimer
MN SGP *
SBP-alpha
PAS-2
Glycophorin C
Component D
SGP-beta
Glycophorin D
Component E
SGP-gamma
PAS-3
Glycophorin B
Ss SGP
SGP-delta
PAS-4
Glycophorin AB heterodimer
MN/Ss
heterodimer
SGP-alpha/delta
heterodimer
*SGP, Sialoglycoprotein.
Depending on the resolution of the gel, the PAS-2 region may contain glycophorin
A monomer, glycophorin C, and glycophorin B homodimer.
201
migrates anomalously, with an apparent molecular weight of 36,000 to 39,000, presumably because of its
highly glycosylated state; approximately 60% of its weight is attributable to carbohydrate. Most of the
carbohydrate is in the form of 15 O-glycosidically linked tetrasaccharides. The two sialic acid residues of
each of these many O-glycosidically linked oligosaccharides account for 60% of the negative charge of the
red cell. The other two components of these tetrasaccharides are one N-acetyl-galactosamine residue and
one galactose residue. In addition, glycophorin A bears one complex N-glycosidically linked
oligosaccharide. Glycophorin A also bears blood group antigens. The N-terminus of glycophorin A bears
the M or N antigen, depending on whether serine and glycine or leucine and glutamic acid are in amino
acid positions one and five, respectively. Although the antigenic polymorphism of the MN system thus
depends on amino acid sequence differences, many human antibodies to these antigens do not recognize
these antigens if sialic acid has been removed from the three O-linked oligosaccharides normally attached
to amino acids two, three, and four. Glycophorin A has also been found to be a binding site for several
pathogens, including Plasmodium falciparum.
[78] [79]
[80]
[81] [82]
[76]
[83]
[84]
[76]
[86]
[87]
[87]
[88]
[89]
[90]
Cytoskeletal Proteins
The most abundant of the peripheral proteins are those that make up
the so-called spectrin-actin cytoskeletal complex. These proteins,
which can be extracted in the presence of EDTA and other chelating
agents, or by reducing ionic strength and raising pH, account for about
35% of the membrane protein. The complex includes large alpha and
beta spectrin polypeptide chains (bands 1 and 2 on gel electrophoresis,
202
molecular weight about 240,000 and 225,000, respectively) and a smaller chain corresponding to band 5.
The term spectrin was originally applied to preparations containing all 3 components but now is used to
refer only to the 2 larger components. The smaller component (band 5) is called erythrocyte actin. The
relationship between the integral and peripheral proteins of the membrane is illustrated in Figure 10.9 .
Study of the erythrocyte cytoskeleton has led to the realization that similar spectrin-based skeletal
structures are important not only in preserving erythrocyte integrity in the face of the shear stresses of the
circulatory system and spleen but also in the function of more complex nucleated cells [91] [92] .
[36]
[36]
[96]
[97]
Figure 10-9 Model of the relationship between integral and cytoskeletal membrane
proteins. Cytoplasmic domains of several integral membrane proteins interact with
several cytoskeletal proteins. (From Gardner K, Bennett GV. Recently identified
erythrocyte membrane-skeletal proteins and interactions. In: Agre P, Parker JC,
editors. Red blood cell membranes--Structure, function, clinical implications. New
203
Figure 10-10 The alpha and beta subunits of human erythrocyte spectrin. Each
protein has numerous repeated segment domains, and the proteins combine to form
antiparallel heterodimers. Most repeat domains (represented as rectangles) are
homologous and are exactly 106 amino acid residues in length; nonhomologous
domains are represented by squares. Roman numerals represent domains identified
historically as numbered from the spectrin self-association site ( at left). Peptides
produced by limited trypsin cleavage, often used to identify the many spectrin
variants associated with abnormal erythrocyte shape, are indicated by " T," followed
by the molecular weight (in thousands) of the fragment. (From Speicher DW. The
present status of erythrocyte spectrin structure: The 106-residue repetitive structure
is a basic feature of an entire class of proteins. J Cell Biochem 30:245, 1986.)
cytoskeleton to the membrane [98] . About 100,000 copies of ankyrin are expressed per cell; at least two
forms of ankyrin, a 206,000 dalton form and a 190,000 dalton form, are present in membranes. The smaller
form has also been designated band 2.2. Both forms contain an amino-terminal domain that binds the anion
exchanger band 3 and a domain more toward the carboxy-terminal that binds spectrin. A peptide within the
carboxy-terminal domain of protein 2.1 is absent from protein 2.2 because of differential processing of
mRNA; this relatively small difference increases the affinity with which ankyrin binds both spectrin and
band 3.
[100] [101]
[103]
[102]
[104]
204
[108]
[109]
[110]
[111]
[114]
[115]
[116]
205
nonmembrane-spanning domains of the alpha subunit are primarily cytoplasmic. In the membrane, the NaK ATPase may exist as oligomers of the alpha, beta, and gamma units in a ratio of 1:1:1.
The erythrocyte also has a urea transporter that transports urea rapidly
across the membrane and helps preserve red cell osmotic stability and
deformability
. Although this transporter has similar characteristics
to the renal urea transporter, they appear to arise from related but
distinct genes
.
[117]
[118] [119]
[121] [122]
[38]
[123]
[122]
Among the enzymes required for the production and use of ATP are
three enzymes that are thought to form a membrane-bound enzyme
complex: aldolase, glyceraldehyde-3 phosphate dehydrogenase (G3PD), and phosphoglycerate kinase. Together, these three enzymes
convert fructose diphosphate to 3-phosphoglycerate with the
production of ATP. G-3PD is the enzyme present in greatest amount in
membrane preparations and is seen as band 6 in polyacrylamide gels.
G-3PD is also found in the erythrocyte cytoplasm and can be
demonstrated to bind to a cytoplasmic segment of band 3. Although
the exact physiologic role of membrane binding for this enzyme
remains unclear, its possible role in regulating metabolic responses to
cell injury and stress is intriguing
.
ATP is not only generated by membrane-bound enzymes, but also is
used by membrane-bound molecules, among which are adenyl cyclase,
which catalyzes the conversion of ATP to cyclic AMP (cAMP), protein
kinases, and adenosine triphosphatases (ATPases). The latter are
involved in several transport functions, as was discussed previously.
Protein kinases are enzymes that phosphorylate other proteins in the
presence of ATP by forming phosphoserine or phosphothreonine bonds.
Phosphorylation is a major step in the regulation of a variety of target
molecules, including structural proteins and enzymes. Erythrocytes
contain numerous protein kinases, including both cytosolic and
membrane-bound cAMP-dependent kinases, cytosolic and membranebound cAMP-independent protein kinases, protein kinase C, and a
calcium-regulated protein kinase. Both membrane-bound and cytosolic
kinases may phosphorylate membrane proteins
. In general,
phosphorylated structural proteins demonstrate lower affinity binding
to their target proteins than do unphosphorylated proteins. For
example, phosphorylation of protein 4.1 leads to a decreased affinity
for spectrin and a decreased ability of 4.1 to promote spectrin- tin
association . Phosphorylation of spectrin, however, leads to little if
any change in spectrin self-association or in association of spectrin
with other molecules, such as ankyrin and actin . Exactly how such
processes regulate cell shape and membrane integrity has not been
well worked out as yet. In the case of protein 4.1, dephosphorylation
because of ATP depletion, an event that might be associated with
stress, would be predicted to lead to a more rigid spectrin-actin
network and reduced membrane deformability
. Phosphorylation of
enzymes can likewise lead to activation of a variety of metabolic
pathways; often, phosphorylation of one molecule leads to activation of
both an enzyme system and the molecules that down-regulate activity
of that system.
Enzymes that use and degrade ATP are also found in the membrane,
although they are not present in large enough quantity to account for
bands seen in Coomassie-stained polyacrylamide gels of membrane
proteins. Like protein kinases, ATPases phosphorylate membrane
[124]
[125]
[36]
[36]
[126]
[127]
exercise, these quantities increase tenfold. If the respiratory gases were carried in physical solution in the
plasma, man's activity would be restricted to only one fiftieth of that possible in the presence of
hemoglobin-containing red cells. Hemoglobin permits the transportation of a hundred times as much
oxygen as could be carried by the plasma alone.
carbon of acetate by
s, those derived from the carboxyl group of acetate by x. The
unmarked carbons are those derived from either the methyl carbon atom of acetate
or from the carboxyl atom. (Prepared by Dr. G.E. Cartwright.)
molecule similar to myoglobin. Such an evolution would explain the high degree of homology between the
alpha and non-alpha ( , gamma, beta, and delta) chains, as well as the extraordinary similarities among
the non-alpha globins. The alpha and non-alpha peptide chains most likely arose because of gene
duplication, after which time the genes for these individual proteins evolved independently. Likewise, the
beta, delta, gamma, and
globins probably also arose as a result of gene duplication. [128] The occurrence
of subunit cooperativity brought with it the advantage of increased physiologic effectiveness of the
hemoglobin molecule and added a new pressure on further evolution of the hemoglobin chains.
[130]
Ontogeny of Hemoglobins
[133]
[135]
[136]
[137]
[138]
207
Designation
Molecular Structure
Adults
Newborns
Adult hemoglobin
alpha2 beta2
97%
20%
Hemoglobin A2
A2
alpha2 delta2
2.5%
0.5%
Fetal hemoglobin
alpha2 gamma2
<1%
80%
Portland
sigma2 gamma2
Gower I
sigma2
Gower II
alpha2
gamma chains are encoded by pairs of genes located near the normal beta chain genes on chromosome 11.
The two genes encode nearly identical proteins: Ggamma has a glycine at codon position where Agamma has an
alanine [139] [140] . In addition, many Agamma genes also encode a threonine-for-isoleucine substitution at position
75 of the protein [141] [142] . During fetal life, Ggamma constitutes about 75% of gamma chains, whereas
hemoglobin F in adults contains about 60% Agamma chains [143] [144] .
neutral buffer
. This property of fetal hemoglobin has been
attributed to amino acid differences in the amino terminus of the
gamma chains that impair binding of 2,3, DPG, an allosteric modifier of
oxygen binding
. Hemoglobin F also has an enhanced alkaline Bohr
effect
.
Late in fetal development, the site of erythropoiesis switches from the
liver and spleen to the bone marrow where progenitors express
predominately adult globins, alpha and beta. However, some beta
globin is expressed during earlier fetal development and may
constitute 5% of beta family globin expression during this time.
Beginning at the 30th week and proceeding to the time of birth, a
significant switch from fetal to adult erythropoiesis takes place, such
that at the time of birth, fetal hemoglobin constitutes approximately
80% of the total hemoglobin. Over the next 25 to 30 weeks, fetal
hemoglobin concentration decreases by approximately 10% every two
weeks until it reaches its normal adult level of less than 2% by 30
weeks of age
. Neonates with hemoglobinopathies or erythropoietic
stress can have a greatly prolonged production of hemoglobin F,
sometimes extending into adulthood
. The proportion of
hemoglobins produced during the different developmental periods is
summarized in Table 10.5 .
Hemoglobin A, alpha2 beta2, is the predominant adult hemoglobin and
normally constitutes approximately 96% of the total adult hemoglobin.
A minor adult hemoglobin, A2 is produced beginning at 35 weeks of
gestation but has little physiologic relevance. Hemoglobin A2 is
composed of alpha globins and the minor adult globin delta. It normally
constitutes less than 3.5% of total adult hemoglobin and its major
clinical importance is its value in diagnosing thalassemias
.
[145]
[146]
[147]
[148]
[149]
[150]
[152]
[153]
1-deoxy fructose
. With special techniques, two different derivatives
can be detected in the AIa fraction. In so-called hemoglobin AIa1 , a
fructose 1,6-diphosphate molecule is attached to the beta chain,
whereas in hemoglobin AIa2 , glucose-6-phosphate occupies the same
site
. Hemoglobin AIb has not yet been fully characterized, but it
appears to be a glycosylated, nonphosphorylated derivative. Levels of
hemoglobins AIa and AIb , like hemoglobin AIc , are also increased in
persons with hyperglycemia.
Because the glycosylated hemoglobins are synthesized throughout the
life span of the red cell, older cells contain a higher proportion of these
variants than younger ones
. Preferential destruction of older cells
explains the observation that the proportion of these hemoglobins is
reduced in hemolytic anemia
. Because the rate of synthesis of
hemoglobins AIa and AIc depends on the blood glucose level; the
concentration of hemoglobin AIc at any one time is proportional to the
average blood sugar over the previous 2 to 3 months
. For this
reason, the level of glycosylated hemoglobins is now used widely as a
measure of glucose control in diabetic patients, as well as a tool for
diagnosis of diabetic states
. In contrast, the hemoglobin AIa
components are not significantly increased in diabetes (Table 10.6) ,
presumably reflecting the fact that their phosphorylated substituents
are not increased in the red cells of patients with diabetes
.
[154]
[152]
[155]
[156]
[157]
[152]
[164]
[168]
[169]
[170]
[171]
Structure of Globin
[172]
[2]
[3]
[4]
[173]
[174]
beta-terminal Group
Normal
Diabetes
AIa1
Fructose 1, 6-diphosphate
0.19
0.2
AIa2
Glucose-6-phosphate
0.19
0.22
AIb *
Unknown
0.48
0.67
AIc
Glucose
3.3
7.5
6.51.5 *
11.02.9 *
Note: Totals are greater than the sum of the components because of differences in technique in the two
studies.
*Mean1 SD, 20 normal subjects and 75 adult diabetics. (Trivelli LA, et al.
Hemoglobin components in patients with diabetes mellitus. N Eng J Med 284:353,
1971.)
209
in the alpha globin chains, many do not assign it a helix designation. The helixes make up about 75% of the
molecule. Interspersed between them are seven nonhelical segments: NA, AB, CD, EF, FG, GH, and HC.
This arrangement is important structurally, because the helixes are relatively rigid and linear, whereas the
nonhelical segments allow bending.
bonding that occurs between them makes the structure stable. The
resulting, roughly spherical, tertiary structure is similar for all the
normal hemoglobin polypeptides (Fig. 10.14) as well as for certain
other heme proteins, such as myoglobin.
The heme pocket is the site of many dynamic interactions involving
oxygen binding to hemoglobin. Heme is suspended in a nonpolar
crevice between the E and F helixes (Fig. 10.14) and helixes B, G, and
H constitute the floor of the pocket. Heme iron forms a covalent bond
with the imidazole nitrogen of the "proximal" histidine at F8. In
addition, heme forms Van der Waals' bonds with many other parts of
the molecule and in this way makes an important contribution to
tertiary structure. If heme is extracted, the central helical regions, C, D,
E, and F, unfold with a consequent decrease in solubility
. Not
surprisingly, some unstable hemoglobins (Chapter 53) result from
amino acid substitutions in the sequences that line the heme pocket
.
The binding of oxygen to the iron molecule causes the hemoglobin
molecule to undergo conformational changes that affect the binding of
oxygen to other heme sites. The mechanism for this property can be
explained in part by the interactions in the heme pocket. The two
histidines of globin are located immediately above and below iron,
which is in the plane of the pyrrole ring in oxyhemoglobin
. In
deoxyhemoglobin, the bond between the imidazole nitrogen of the
proximal histidine and iron undergoes considerable strain, displacing
iron from the plane of the ring. This strain is in part responsible for the
T or tense state of deoxyhemoglobin
. The addition of two molecules
of oxygen, which is bound to the iron atom in the heme ring by end-on
geometry, results in the formation of a hydrogen bond between the
oxygen atom not bound directly to the iron and the imidazole nitrogen
of the histidine at E7 (the "distal" histidine)
. The binding of oxygen
to iron changes the electron spin state of iron and relaxes the covalent
bond with the proximal histidine, permitting the iron to move into the
plane of the ring and relaxing the molecule, contributing to the R or
relaxed state
. The overall conformational changes to hemoglobin
appear to be the greatest after three molecules of O2 have been added.
When four polypeptide chains combine to form the hemoglobin
molecule, each chain lies approximately at the vertices of a regular
tetrahedron. With high resolution x-ray diffraction, the nature of the
contacts between chains has been explored in detail for horse
hemoglobin
. Contacts between like chains, i.e., alpha1 alpha2 and
beta1 beta2 , are limited and of little importance. The two major
contacts between unlike chains have been named alpha1 beta1 and
alpha1 beta2 , respectively (Fig. 10.15) . (The alpha2 beta2 contact is the
same as alpha1 beta1 .) The alpha1 beta1 contact point is extensive and
moves relatively little (less than 0.1 nm) when hemoglobin is
oxygenated. The alpha1 beta2 contact is smaller and smoother, and
[176]
[177]
[178]
[179]
[179]
[180]
[174]
[182]
[183]
[184]
210
alpha
beta
gamma
delta
NA1
Val
Val
Gly
Val
NA2
Leu
His
His
His
Leu
Phe
Leu
Thr
Thr
Thr
NA3
A1
Ser
alpha
beta
gamma
delta
A2
Pro
Pro
Glu
Pro
A3
Ala
Glu
Glu
Glu
A4
Asp
Glu
Asp
Glu
A5
Lys
Lys
Lys
Lys
A6
Thr
Ser
Ala
Thr
A7
Asn
Ala
Thr
Ala
10
A8
10
Val
Val
Ile
Val
11
A9
11
Lys
Thr
Thr
Asn
12
A10
12
Ala
Ala
Ser
Ala
13
A11
13
Ala
Leu
Leu
Leu
14
A12
14
Try
Try
Try
Try
15
A13
15
Gly
Gly
Gly
Gly
16
A14
16
Lys
Lys
Lys
Lys
17
A15
17
Val
Val
Val
Val
18
A16
18
Gly
AB1
19
Ala
B1
20
His
Asn
Asn
Asn
19
B2
21
Ala
Val
Val
Val
20
B3
22
Gly
Asp
Glu
Asp
21
B4
23
Glu
Glu
Asp
Ala
22
B5
24
Tyr
Val
Ala
Val
23
B6
25
Gly
Gly
Gly
Gly
24
B7
26
Ala
Gly
Gly
Gly
25
B8
27
Glu
Glu
Glu
Glu
26
B9
28
Ala
Ala
Thr
Ala
27
B10
29
Leu
Leu
Leu
Leu
28
B11
30
Glu
Gly
Gly
Gly
29
B12
31
Arg
Arg
Arg
Arg
30
B13
32
Met
Leu
Leu
Leu
31
B14
33
Phe
Leu
Leu
Leu
32
alpha
beta
gamma
delta
B15
34
Leu
Val
Val
Val
33
B16
35
Ser
Val
Val
Val
34
C1
36
Phe
Tyr
Tyr
Tyr
35
C2
37
Pro
Pro
Pro
Pro
36
C3
38
Thr
Try
Try
Try
37
C4
39
Thr
Thr
Thr
Thr
38
C5
40
Lys
Gln
Gln
Gln
39
C6
41
Thr
Arg
Arg
Arg
40
C7
42
Tyr
Phe
Phe
Phe
41
CD1
43
Phe
Phe
Phe
Phe
42
CD2
44
Pro
Glu
Asp
Glu
43
CD3
45
His
Ser
Ser
Ser
44
CD4
46
Phe
Phe
Phe
Phe
45
CD5
47
Asp
Gly
Gly
Gly
46
CD6
48
Leu
Asp
Asn
Asp
47
CD7
49
Ser
Leu
Leu
Leu
48
Ser
Ser
Ser
49
CD8
D1
50
His
Thr
Ser
Ser
50
D2
51
Gly
Pro
Ala
Pro
51
D3
Asp
Ser
Asp
52
D4
Ala
Ala
Ala
53
D5
Val
Ile
Val
54
D6
Met
Met
Met
55
D7
Gly
Gly
Gly
56
E1
52
Ser
Asn
Asn
Asn
57
E2
53
Ala
Pro
Pro
Pro
58
E3
54
Gln
Lys
Lys
Lys
59
E4
55
Val
Val
Val
Val
60
E5
56
Lys
Lys
Lys
Lys
61
E6
57
Gly
Ala
Ala
Ala
62
alpha
beta
gamma
delta
E7
58
His
His
His
His
63
E8
59
Gly
Gly
Gly
Gly
64
E9
60
Lys
Lys
Lys
Lys
65
E10
61
Lys
Lys
Lys
Lys
66
E11
62
Val
Val
Val
Val
67
E12
63
Ala
Leu
Leu
Leu
68
E13
64
Asp
Gly
Thr
Gly
69
E14
65
Ala
Ala
Ser
Ala
70
E15
66
Leu
Phe
Leu
Phe
71
E16
67
Thr
Ser
Gly
Ser
72
E17
68
Asn
Asp
Asp
Asp
73
E18
69
Ala
Gly
Ala
Gly
74
E19
70
Val
Leu
Ile
Leu
75
E20
71
Ala
Ala
Lys
Ala
76
EF1
72
His
His
His
His
77
EF2
73
Val
Leu
Leu
Leu
78
EF3
74
Asp
Asp
Asp
Asp
79
EF4
75
Asp
Asn
Asp
Asn
80
EF5
76
Met
Leu
Leu
Leu
81
EF6
77
Pro
Lys
Lys
Lys
82
EF7
78
Asn
Gly
Gly
Gly
83
EF8
79
Ala
Thr
Thr
Thr
84
F1
80
Leu
Phe
Phe
Phe
85
F2
81
Ser
Ala
Ala
Ser
86
F3
82
Ala
Thr
Gln
Gln
87
F4
83
Leu
Leu
Leu
Leu
88
F5
84
Ser
Ser
Ser
Ser
89
F6
85
Asp
Glu
Glu
Glu
90
F7
86
Leu
Leu
Leu
Leu
91
F8
87
His
His
His
His
92
alpha
beta
gamma
delta
F9
88
Ala
Cys
Cys
Cys
93
FG1
89
His
Asp
Asp
Asp
94
FG2
90
Lys
Lys
Lys
Lys
95
FG3
91
Leu
Leu
Leu
Leu
96
FG4
92
Arg
His
His
His
97
FG5
93
Val
Val
Val
Val
98
G1
94
Asp
Asp
Asp
Asp
99
G2
95
Pro
Pro
Pro
Pro
100
G3
96
Val
Glu
Glu
Glu
101
G4
97
Asn
Asn
Asn
Asn
102
G5
98
Phe
Phe
Phe
Phe
103
G6
99
Lys
Arg
Lys
Arg
104
G7
100
Leu
Leu
Leu
Leu
105
G8
101
Leu
Leu
Leu
Leu
106
G9
102
Ser
Gly
Gly
Gly
107
G10
103
His
Asn
Asn
Asn
108
G11
104
Cys
Val
Val
Val
109
G12
105
Leu
Leu
Leu
Leu
110
G13
106
Leu
Val
Val
Val
111
G14
107
Val
Cys
Thr
Cys
112
G15
108
Thr
Val
Val
Val
113
G16
109
Leu
Leu
Leu
Leu
114
G17
110
Ala
Ala
Ala
Ala
115
G18
111
Ala
His
Ile
Arg
116
G19
112
His
His
His
Asn
117
GH1
113
Leu
Phe
Phe
Phe
118
GH2
114
Pro
GLy
Gly
Gly
119
GH3
115
Ala
Lys
Lys
Lys
120
GH4
116
Glu
Glu
Glu
Glu
121
GH5
117
Phe
Phe
Phe
Phe
122
alpha
beta
gamma
delta
H1
118
Thr
Thr
Thr
Thr
123
H2
119
Pro
Pro
Pro
Pro
124
H3
120
Ala
Pro
Glu
Gln
125
H4
121
Val
Val
Val
Met
126
H5
122
His
Gln
Gln
Gln
127
H6
123
Ala
Ala
Ala
Ala
128
H7
124
Ser
Ala
Ser
Ala
129
H8
125
Leu
Tyr
Tyr
Tyr
130
H9
126
Asp
Gln
Gln
Gln
131
H10
127
Lys
Lys
Lys
Lys
132
H11
128
Phe
Val
Met
Val
133
H12
129
Leu
Val
Val
Val
134
H13
130
Ala
Ala
Thr
Ala
135
H14
131
Ser
Gly
H15
132
Val
Val
Val
Val
137
H16
133
Ser
Ala
Ala
Ala
138
H17
134
Thr
Asn
Ser
Asn
139
H18
135
Val
Ala
Ala
Ala
140
H19
136
Leu
Leu
Leu
Leu
141
H20
137
Thr
Ala
Ser
Ala
142
H21
138
Ser
His
Ser
His
143
HC1
139
Lys
Lys
Arg
Lys
144
HC2
140
Tyr
Tyr
Tyr
Tyr
145
HC3
141
Arg
His
His
His
146
Gly
Gly
136
*Amino acids are indicated by a three-letter code. Uncharged amino acids: Gly, glycine; Ala, alanine; Val,
valine; Leu, leucine; Ile, isoleucine; Gln, glutamine; Asn, asparagine; Met, methionine; Cys, cystine; Phe,
phenylalanine; Tyr, tyrosine; Try, tryptophan; Pro, proline; Ser, serine; Thr, threonine. Charged amino
acids: Asp, aspartic acid (-1); Glu, glutamic acid (-1); Arg, arginine (+1); Lys, lysine (+1); His, histidine
(+1).
The two normal gamma-chain genes differ from each other in that the G gamma gene encodes glycine at
this position, whereas the A gamma gene encodes alanine.
vitro mixing experiments [183] . Conversely, more negatively charged variants such a betaN-Baltimore bind with a
greater association rate. This phenomenon has be suggested as an explanation for the ratio of 60:40 seen for
hemoglobin A and hemoglobin S in heterozygotes for betaS . Likewise the percentage of N-Baltimore is
increased over hemoglobin A.
[186]
Oxygen transport
In order to function as the primary medium of exchange of oxygen and
carbon dioxide, hemoglobin must fulfill the four requirements first
delineated by Barcroft
. It must be capable of transporting a large
quantity of oxygen, it must be highly soluble, it must take up and
release oxygen at "appropriate pressures," and it must also be a good
buffer. Normal hemoglobin fulfills these requirements well, although
many abnormal variants fail to meet one or more of these conditions.
When fully saturated, each gram of hemoglobin binds 1.39 mL of
oxygen. The degree of saturation is related to the oxygen tension (P
O2 ), which normally ranges from 100 mm Hg in arterial blood to about
35 mm Hg in veins. The relation between oxygen tension and
hemoglobin oxygen saturation is described by the oxygen-dissociation
curve of hemoglobin (Fig. 10.17) . The characteristics of this curve are
related in part to properties of hemoglobin itself and in part to the
environment within the erythrocyte, with pH, temperature,
[187]
Figure 10-14 The tertiary structure of a single globin polypeptide chain. The helical
segments, labeled A through H, are relatively linear; bending of the chains occurs
between helices. Heme is suspended in a crevice between the E and F helices.
(Courtesy of C. A. Finch.)
212
Figure 10-15 Diagram of hemoglobin tetramer illustrates two types of alphabetacontact points: A relatively extensive one (alpha1 beta1 or alpha2 beta2 ) and one that
is smaller (alpha1 beta2 or alpha2 beta1 ). The actual molecular contact points are
[189]
[190]
213
[200]
[202]
[206]
Figure 10-18 The synthesis of 2,3-diphosphoglycerate ( 2,3-DPG) or 3phosphoglycerate ( 3-PG) and ATP from 1,3-diphosphoglycerate ( 1,3-DPG) by the
Rapoport-Luebering cycle. (From Bunn HF, Forget BG. Hemoglobin: Molecular, genetic
and clinical aspects. Philadelphia, WB Saunders, 1986, with permission.)
214
is not essential to life, however; an individual who lacked the enzymes necessary for 2,3-DPG synthesis
was perfectly well except for mild polycythemia. Heme-heme interaction, the Bohr effect, and the effect of
2,3-DPG have been explained on a molecular basis in a model proposed by Perutz [207] . In the completely
deoxygenated state, hemoglobin assumes a quaternary structure termed T ("taut" or "tense"). This structure
is stabilized by salt bridges involving the carboxy terminals of the peptide chains. The deoxy form is also
stabilized by the presence of 2,3-DPG, which joins the beta chains as shown in Figure 10.19 .
delineated
. Hemoglobin appears to exist in a third molecular
form, intermediate between the T and R conformations. Achievement
of the R conformation occurs when at least one oxygen molecule is
[208] [209]
Figure 10-19 The 2,3-DPG binding site. Part of one beta chain is at upper left, the
other at lower right. Salt bridges are found between the phosphates of 2,3-DPG and
positively charged groups at 1 Val (a-NH2+ ), 2 His, and 143 His. The 82 Lys of one
chain is also involved. (From Arnone A. X-ray diffraction study of binding of 2,3diphosphoglycerate to human deoxyhemoglobin. Nature 237:146, 1972, with
permission.)
215
diffuses freely across the red cell membrane and a portion is exchanged with plasma Cl- , a phenomenon
called the "chloride shift." The bicarbonate is carried in plasma to the lungs, where ventilation keeps the P
CO2 low, resulting in reversal of the above reactions and excretion of CO2 in the expired air. About 85% of
tissue carbon dioxide is processed in this way and 5% is carried in simple solution.
Figure 10-21 The carbon dioxide dissociation curve of whole blood. The curve is
relatively linear between pCO2 40 and 60 mm Hg. The difference between the
curves of oxygenated and deoxygenated blood is known as the Haldane effect.
[219] [220]
[221] [222]
[223]
[224]
[225]
216
[226]
[227]
217
Formula
Hb(d6 2 )
5
lambda
lambda
lambda
e
--
430
133
555
12.5
--
Oxyhemoglobin
Hb(d -
415
125
541
13.8
577
14.6
Acid methemoglobin
405
179
500
10.0
631
4.4
Alkaline methemoglobin
410
120
540
11.0
575
9.2
Cyanomethemoglobin
Hb(d5 566)S
421
86
618
24
and, in concentrations greater than 10% of the total hemoglobin, imparts to blood a distinctive brownish
hue that does not disappear on vigorous shaking in air [228] . When methemoglobin is present in vivo in
concentrations greater than 1.5 to 2.0 g/dl, patients appear visibly cyanotic. Methemoglobin combines
readily with cyanide to form cyanomethemoglobin, a pigment so stable that it is used in laboratory
procedures for quantifying hemoglobin.
[229]
218
is sulfhemoglobin. It is a relatively stable pigment and, once formed, cannot be converted to hemoglobin in
vivo. Instead, it tends to remain within the cell throughout the cell's life. Sulfhemoglobin is bright green
and has a distinctive spectrum characterized by an absorption band at about 618 nm (Table 10.7) . It is a
ferrous compound with one sulfur atom attached to each heme group. The sulfur is probably attached to a
beta-carbon in the porphyrin ring, forming a thiochlorin (Fig. 10.25) [230] [231] .
[232]
Methemoglobin Reduction
[234]
Cytochrome b5 reductase
67%
Ascorbic acid
16%
Glutathione
12%
5%
Total
100%
[239]
[240]
219
[243] [244]
[245]
[246] [247]
[248]
[243]
[244]
[249]
[246] [247]
[250] [251]
220
[253] [254]
exemplified by the fact that a genetic defect in the enzyme may lead
to a drug-sensitive hemolytic anemia
.
Catalase, a heme enzyme, decomposes hydrogen peroxide to water
and molecular oxygen
. It appears to be less important to the red
cell than peroxidase, presumably because it is effective only when the
peroxide concentration is relatively high
. Individuals with hereditary
acatalasemia do not develop methemoglobinemia or hemolytic
disease; an increase in glutathione peroxidase levels may compensate
in part for the lack of catalase
. Some evidence suggests, however,
that erythrocyte catalase may be important in preventing oxidant
damage to somatic tissues
. Also, the level of catalase increases
with physical conditioning, suggesting a physiologically significant role
for erythrocyte catalase
.
Catalase consists of a tetramer composed of 60,000-dalton subunits,
with four heme groups per tetramer. It is encoded by a gene on
chromosome 11
. Catalase is a major component of erythrocyte
band 4.5 seen on Coomassie-stained gels of erythroid membrane
proteins, as the enzyme interacts with the membrane in a calcium- and
pH-dependent manner
. Catalase also comprises a major reservoir of
erythrocyte protein-bound NADPH. Each tetrameric molecule of
erythrocyte catalase contains four molecules of tightly bound NADPH.
Although not essential for enzymatic conversion of peroxide to oxygen,
the NADPH appears to protect catalase from inactivation by peroxide
.
[255]
[256]
[257]
[258]
[259]
[260]
[261]
[262]
[263]
Glutathione Metabolism
Glutathione is the principal reducing agent in erythrocytes and the
essential cofactor in the glutathione peroxidase reaction. Reduced
glutathione (GSH) is a tripeptide (gamma-glutamyl-cysteinyl-glycine).
Two ATP-dependent enzymatic reactions are required for the de novo
synthesis of glutathione:
1. glutamic acid + cysteine gamma-glutamyl-cysteine
2. gamma-glutamyl-cysteine + glycine GSH
Reaction 1 is catalyzed by glutamyl-cysteine synthetase, reaction 2 by
glutathione synthetase. Both reactions can take place in normal
erythrocytes
. The capacity of normal red cells to synthesize
glutathione exceeds the rate of turnover by 150-fold.
In the course of reactions protecting hemoglobin from oxidation, GSH is
oxidized, forming oxidized glutathione (GSSG), which consists of two
GSH molecules joined by a disulfide linkage, and mixed disulfides with
hemoglobin. GSSG rapidly leaves the erythrocyte
. Thus, maintaining
a continuous supply of GSH requires a system to reduce the oxidized
forms of glutathione. Such a system is provided by glutathione
reductase, which catalyzes the reduction of GSSG by NADPH, a product
of the pentose phosphate pathway. (Fig. 10.27) . Glutathione reductase
[264]
[265]
[270] [271]
[272]
Figure 10-28 Energy metabolism in the erythrocyte. Main pathways are shown
as boxes; major substrates and products of each are shown outside the boxes.
More details of the pathways are given in Figures 10.27 and 10.29 . Hb,
hemoglobin; MHb, methemoglobin; NAD, NADH, nicotinamide adenine dinucleotide;
ADP, adenosine diphosphate; ATP, adenosine triphosphate; NADP, NADPH,
nicotinamide adenine dinucleotide phosphate; G-6-P, glucose-6-phosphate; F-6-P,
fructose-6-phosphate; Ga-3-P, glyceraldehyde-3-phosphate; 2,3 DPG, 2,3diphosphoglycerate.
221
Energy Metabolism
Although the mature red cell contains the enzymes required for
glycogen metabolism, the balance between synthesis and utilization is
such that no significant amount of glycogen accumulates within the
cell under normal circumstances
. Glycogen may accumulate,
however, in glycogen storage diseases types III and VI.
Lacking a storage compound, the normal erythrocyte must have
constant access to glucose if its energy metabolism is to be sustained.
As previously discussed, glucose enters the cell by means of a
facilitated, carrier-mediated transport mechanism. Insulin or other
hormones are not required, and transport is not ordinarily the ratelimiting factor in glucose utilization. Without mitochondria,
erythrocytes must depend on two less efficient pathways for
production of high-energy compounds, the anaerobic glycolytic
(Embden-Meyerhof) pathway and the aerobic pentose phosphate
pathway, also known as the hexose monophosphate shunt or the
phosphogluconate pathway (Fig. 10.28) . Under normal circumstances,
about 90% of glucose entering the red cell is metabolized by the
anaerobic pathway and 10% by the aerobic pathway
. Under
conditions of oxidative stress, however, the oxidative pentose pathway
may account for up to 90% of glucose consumption
.
Three important products are formed by the anaerobic glycolytic
pathway: NADH, a cofactor in the methemoglobin reductase reaction;
ATP, the major high-energy phosphate nucleotide that powers the
cation pump; and 2,3-DPG, a regulator of hemoglobin function (Fig.
10.28) . For each molecule of glucose that enters the pathway, two
molecules of NADH are generated (Fig. 10.29 , reaction 6). The yields
of ATP and 2,3-DPG vary depending on the activity of the RapoportLuebering shunt (Fig. 10.29 , reactions 7b and 7c), a side pathway
unique to the red cell. Two molecules of ATP are used in the early steps
of glycolysis (Fig. 10.29 , reactions 1 and 3) and a maximum of four
molecules is produced late
[274]
[275]
[276]
222
in the pathway (two in reaction 7a and two in reaction 10). Thus, at maximum efficiency, a net yield of two
molecules of ATP may be expected for each molecule of glucose catabolized. This net yield may be
decreased, however, to the extent that 2,3-DPG is formed (Fig. 10.29 , reactions 7b and 7c). For this reason,
the DPG-forming step is sometimes referred to as an energy clutch.
Activity *
Hexokinase (HK)
1.3
61.0
Phosphofructokinase (PFK)
9.0
Aldolase (Ald)
3.2
2111.0
Glyceraldehyde-3-phosphate dehydrogenase
(GAPD)
226.0
320.0
Diphosphoglyceromutase (DPGM)
4.8
Phosphoglyceromutase (PGM)
25.0
Enolase
5.4
15.0
200.0
*Micromoles per minute per gram of hemoglobin at 37C and at high substrate
concentrations.
methylene blue, cysteine, ascorbate, and others induce an up to 20-fold increase in pentose metabolism,
presumably by bringing about oxidation of glutathione [279] [280] . This metabolic flexibility allows the red cell
to respond to unexpected oxidant challenge.