MODULE 3 Flashcards
is the most common complex organic molecule in vertebrates.
Hemoglobin (Hgb or Hb)
It comprises approximately 95% of the cytoplasmic content of RBCs.
Hemoglobin (Hgb or Hb)
provides protection from denaturation in the plasma and loss through the kidneys
hemoglobin in RBCs
concentration of hemoglobin within RBCs
34 g/dL
molecular weight of hemoglobin within RBCs
64,000 Daltons
Hemoglobin’s main function is to transport oxygen from the (?) and transport carbon dioxide from the (?) for exhalation.
lungs to tissues
tissues to the lungs
Hemoglobin also contributes to (?) by binding and releasing hydrogen ions and transports nitric oxide (NO)
acid-base balance
a regulator of vascular tone
nitric oxide (NO),
Components of Hemoglobin
- Four heme molecules each composed of:
a. The nitrogenous substance, protoporphyrin IX
b. Iron atom in the ferrous (Fe2+) state. - The protein component known as globin made up of two sets or dimers of two different polypeptide chains.
- The transient resident, 2,3-biphosphoglycerate (2,3-BPG) which regulates oxygen affinity to the hemoglobin molecule
- Four heme molecules each composed of:
a. The nitrogenous substance, (?)
b. Iron atom in the (?) state.
protoporphyrin IX
ferrous (Fe2+)
- The protein component known as (?) made up of two sets or dimers of two different polypeptide chains.
globin
- The transient resident, (?) which regulates oxygen affinity to the hemoglobin molecule
2,3-biphosphoglycerate (2,3-BPG)
Structure of the Hemoglobin Components
- Heme molecule
- Globin molecule
- The Complete Hemoglobin Molecule
consists of a ring of carbon, hydrogen, and nitrogen atoms called protoporphyrin IX, with a central atom of divalent ferrous iron (Fe2+)
Heme
Each of the (?) is positioned in a pocket of the polypeptide chain near the surface of the hemoglobin molecule.
four heme groups
The (?) in each heme molecule reversibly combines with one oxygen molecule.
ferrous iron
When the ferrous irons are oxidized to the ferric state (Fe3+) the hemoglobin will become (?), which cannot bind oxygen.
methemoglobin
The (?) comprising each hemoglobin molecule consist of two identical pairs of unlike polypeptide chains, 141 to 146 amino acids each.
four globin chains
Variations in amino acid sequences give rise to different types of
polypeptide chains.
Each chain is designated by a
Greek letter
The hemoglobin molecule can be described by its (?) structures.
primary, secondary, tertiary, and quaternary protein
refers to the amino acid sequence of the polypeptide chains.
primary structure
refers to chain arrangements in helices and non-helices
secondary structure
refers to the arrangement of the helices into a pretzel-like configuration
tertiary structure
loop to form a cleft pocket for heme
Globin chains
Each globin chain contains a heme group that is suspended between the (?) of the polypeptide chain
E and F helices
The (?) at the center of the protoporphyrin IX ring of heme is positioned between two histidine radicals.
iron atom
Globin chain amino acids in the (?) are hydrophobic
cleft
Globin chain amino acids on the (?) are hydrophilic, which renders the molecule water soluble.
outside
This arrangement also helps iron remain in its (?) form regardless of whether it is oxygenated or deoxygenated
divalent ferrous
(carrying an oxygen molecule)
oxygenated
(not carrying an oxygen molecule).
deoxygenated
also called a tetramer; describes the complete hemoglobin molecule.
quaternary structure
The complete hemoglobin molecule is (?), has four heme groups attached to four polypeptide chains, and may carry up to four molecules of oxygen
spherical
The predominant adult hemoglobin, (?) (also known as Hb A), is composed of two α-globin chains and two β-globin chains.
Hb A1
hold the dimers in a stable form
Strong α1–β1 and α2–β2 bonds
are important for the stability of the quaternary structure in the oxygenated and deoxygenated forms
α1–β2 and α2–β1 bonds
Alpha
Beta
Gamma A
Gamma G
Delta
Epsilon
Seta
Theta
This is a substance produced in the anaerobic glycolytic (Embden-Meyerhof) pathway.
2,3-Biphosphoglycerate (2,3-BPG)
This pathway generates energy for red blood cells.
Embden-Meyerhof
is specifically produced in the by-pass pathway within the Embden Meyerhof pathway which is known as the Luebering-Rapoport Shunt.
2,3-BPG
O2 affinity decreases
Hgb binds 2,3-BPG
oxygen affinity increases
Hgb releases the 2,3-BPG
This illustrates the reverse relationship between the amount of 2,3-BPG and the affinity of Hgb for O2.
Luebering-Rapoport Shunt
How is Hemoglobin synthesized by the red blood cells?
A. Heme Biosynthesis
B. Globin Biosynthesis
C. Hemoglobin Assembly (Heme + Globin)
D. Development of Hemoglobin from fetal to adult life (Switching of globin chains)
The biosynthesis of heme occurs mainly in the (?) of the bone marrow red cell precursors starting from the pronormoblast through the circulating reticulocytes.
mitochondria and the cytoplasm
As the red cell further matures and lose their (?), they lose their ability to further synthesize hemoglobin.
ribosomes and mitochondria
Heme biosynthesis begins in the mitochondria with the condensation of (?) catalyzed by aminolevulinate synthase to form (?)
glycine and succinyl coenzyme A (CoA)
aminolevulinic acid (ALA)
In the cytoplasm, ALA undergoes several transformations from (?) to (?), which, catalyzed by (?), becomes (?).
porphobilinogen
coproporphyrinogen III
coproporphyrinogen oxidase
protoporphyrinogen IX
In the mitochondria, (?) is converted to (?) by (?).
protoporphyrinogen IX
protoporphyrin IX
protoporphyrinogen oxidase
(?) is added, catalyzed by (?) to form heme.
Ferrous (Fe2+) ion
ferrochelatase
In the cytoplasm, heme assembles with an α chain and non-a chain, forming a dimer, and ultimately two dimers join to form the
hemoglobin tetramer
(?), a plasma protein, carries iron in the ferric (Fe3+) form to developing erythroid cells.
Transferrin
(?) binds to transferrin receptors on erythroid precursor cell membranes and the receptors and transferrin (with bound iron) are brought into the cell in an endosome.
Transferrin
releases the iron from transferrin
Acidification of the endosome
Iron is transported out of the endosome and into the mitochondria where it is reduced to the ferrous state, and is united with (?) to make heme.
protoporphyrin IX
Heme leaves the mitochondria and is joined to the
globin chains in the cytoplasm
code for six globin chains
Six structural genes
are on the short arm of chromosome 16
α- and ζ-globin genes
is on the short arm of chromosome 11
ε-, γ-, δ-, and β-globin gene cluster
In the human genome, there is one copy of each globin gene per chromatid, for a total of (?), with the exception of a and g.
two genes per diploid cell
There are two copies of the a- and γ-globin genes per chromatid, for a total of
four genes per diploid cell
The production of globin chains takes place in (?) from the pronormoblast through the circulating polychromatic erythrocyte, but not in the mature erythrocyte.
erythroid precursors
Transcription of the globin genes to messenger ribonucleic acid (mRNA) occurs in the
nucleus
translation of mRNA to the globin polypeptide chain occurs on
ribosomes in the cytoplasm
Although transcription of the a-globin genes produces more mRNA than the b-globin gene, there is less efficient translation of the
a-globin mRNA
are produced in approximately equal amounts
a and b chains
After translation is complete, the chains are released from the (?) in the cytoplasm.
ribosomes
After their release from ribosomes, each globin chain binds to a heme molecule, then forms a
heterodimer
have a charge difference that determines their affinity to bind to the α chains.
non-a chains
has a positive charge and has the highest affinity for a β chain due to its negative charge
α chain
has the next highest affinity, followed by the δ-globin chain
γ-globin chain
Two heterodimers then combine to form a (?)
This completes the hemoglobin molecule.
tetramer
Two a and two β chains form (?), the major hemoglobin present from 6 months of age through adulthood
Hb A
contains two a and two δ chains
Hb A2
Owing to a mutation in the promoter region of the δ-globin gene, production of the (?) is very low.
δ chain polypeptide
comprises less than 3.5% of total hemoglobin in adults
Hb A2
contains two a and two γ chains
Hb F
In healthy adults, (?) comprises 1% to 2% of total hemoglobin, and it is present only in a small proportion of the RBCs (uneven distribution).
Hb F
These RBCs with Hb F are called
F or A/F cells
The various amino acids that comprise the globin chains affect the net charge of the
hemoglobin tetramer
are used for fractionation, presumptive identification, and quantification of normal hemoglobin and hemoglobin variants
Electrophoresis and high-performance liquid chromatography (HPLC)
(?) of globin gene DNA provides definitive identification of variant hemoglobin.
Molecular genetic testing
Hemoglobin composition differs with
prenatal gestation time and postnatal age
Hemoglobin changes reflect the sequential activation and inactivation (or switching) of the globin genes, progressing from the (?) on chromosome 16 and from the (?) on chromosome 11.
ζ- to the a-globin gene
ε- to the γ-, δ-, and β-globin genes
normally appear only during the first 3 months of embryonic development.
ζ- and ε-globin chains
These two chains, when paired with the a and γ chains, form the
embryonic hemoglobins
During the second and third trimesters of fetal life and at birth, (?) is the predominant hemoglobin.
Hb F (a2γ2)
By 6 months of age and through adulthood, (?) is the predominant hemoglobin, with small amounts of Hb A2 (a2δ2) and Hb F.
Hb A (a2β2)
In utero, (?) predominates.
fetal hemoglobin
When compared with adult hemoglobin, fetal hemoglobin has (?), a characteristic that allows sufficient oxygen transfer to the fetus in the absence of gas exchange with the external environment due to the relatively hypoxic environment in utero.
very high oxygen-binding capacity
As a result, the hemoglobin level in a near-term fetus or term infant is relatively high and remains elevated up to around the (?) to compensate for the high oxygen affinity of hemoglobin.
8th to the 12th week post-partum
reference intervals for hemoglobin concentration
Men:
Men: 14 to 18 g/dL (140 to 180 g/L)
reference intervals for hemoglobin concentration
Women:
Women: 12 to 15 g/dL (120 to 150 g/L)
reference intervals for hemoglobin concentration
Newborns:
Newborns: 16.5 to 21.5 g/dL (165 to 215 g/L)
Reference intervals for infants and children vary according to
age group
Individuals living at high altitudes have (?) as a compensatory mechanism to provide more oxygen to the tissues in the oxygen-thin air.
slightly higher levels of hemoglobin
Hemoglobin variants are a part of the
normal embryonic and fetal development
They may also be (?) of hemoglobin in a population, caused by variations in genetics.
pathologic mutant forms
Some well-known hemoglobin variants, such as in sickle-cell anemia, are responsible for diseases and are considered (?).
hemoglobinopathies
Other variants cause no detectable pathology, and are thus considered
non-pathological variants
- In the embryo (products of yolk sac erythroblasts)
• Gower 1 (ζ2ε2)
• Gower 2 (α2ε2)
• Hemoglobin Portland I (ζ2γ2)
• Hemoglobin Portland II (ζ2β2)
- In the fetus: (begins in early embryogenesis, peaks during third trimester and declines just before birth)
• Hemoglobin F (α2γ2).
- Right after birth up to before the first year of life:
• Hemoglobin F (α2γ2) at 60 – 90% of total Hb
• Hemoglobin A (adult hemoglobin) (α2β2) at 10 – 40% of total Hb.
- Two years through adulthood:
• Hemoglobin A (adult hemoglobin) (α2β2)
• Hemoglobin A2 (α2δ2)
• Hemoglobin F (fetal hemoglobin) (α2γ2)
– 90% of total Hb
• Hemoglobin F (α2γ2) at 60
– 40% of total Hb.
• Hemoglobin A (adult hemoglobin) (α2β2) at 10
– The most common with a normal amount over 95%
• Hemoglobin A (adult hemoglobin) (α2β2)
– δ chain synthesis begins late in the third trimester and, in adults, it has a normal range of 1.5–3.5%
• Hemoglobin A2 (α2δ2)
– In adults Hemoglobin F is restricted to a limited population of red cells called F-cells (1 – 2%)
• Hemoglobin F (fetal hemoglobin) (α2γ2)
B. Variant forms that may cause disease:
- Hemoglobin D-Punjab (α2βD2)
- Hemoglobin H (β4)
- Hemoglobin Barts (γ4)
- Hemoglobin S (α2βS2)
- Hemoglobin C (α2βC2)
- Hemoglobin E (α2βE2)
- Hemoglobin AS
- Hemoglobin SC
A. Normal Hemoglobins
is one of the sub-variants of Hemoglobin D, a variant of hemoglobin found in human blood
Hemoglobin D-Punjab (α2βD2)
It is so named because of its higher prevalence in the Punjab region of India and Pakistan.
Hemoglobin D-Punjab (α2βD2)
accounts for over 55% of the total hemoglobin variants there
Hemoglobin D-Punjab
Hemoglobin D-Punjab was first discovered in the early 1950s in a mixed British and American family of Indian origin from the Los Angeles area; hence it is also sometimes called
“D Los Angeles”
A variant form of hemoglobin, formed by a tetramer of β chains, which may be present in variants of α thalassemia.
Hemoglobin H (β4)
Although each of the beta (β) globin chains is normal, the (?) does not function normally.
tetramer of 4 beta chains
It has an increased affinity for oxygen, holding onto it instead of releasing it to the tissues and cells.
tetramer of 4 beta chains
is also associated with significant breakdown of red blood cells (hemolysis) as it is unstable and tends to form solid structures within red blood cells
Hemoglobin H
Serious medical problems are not common in people with (?), though they often have anemia.
hemoglobin H disease
– A variant form of hemoglobin, formed by a tetramer of γ chains, which may be present in variants of α thalassemia.
Hemoglobin Barts (γ4)
If a small amount of Hb Barts is detected, it usually disappears shortly after birth due to
dwindling gamma chain production
These children have one or two alpha gene deletions and are silent carriers or have the alpha thalassemia trait.
Hemoglobin Barts (γ4)
If a child has a large amount of Hb Barts, he or she usually has
hemoglobin H disease and a three-gene deletion
have hydrops fetalis and usually do not survive without blood transfusions and bone marrow transplants
Fetuses with four-gene deletions
– A variant form of hemoglobin found in people with sickle cell disease.
Hemoglobin S (α2βS2)
There is a variation in the β-chain gene, causing a change in the properties of hemoglobin, which results in sickling of red blood cells.
Hemoglobin S (α2βS2)
The gene defect is a single nucleotide mutation of the β-globin gene, which results in (?) being substituted by (?) at position 6 (E6V) substitution.
glutamic acid (E/Glu)
valine (V/Val)
This is normally a benign mutation, causing no apparent effects on the secondary, tertiary, or quaternary structures of hemoglobin in conditions of normal oxygen concentration.
Hemoglobin S (α2βS2)
However, under low oxygen concentration, (?) polymerizes and forms fibrous precipitates because the deoxy form of hemoglobin exposes a hydrophobic patch on the protein between the E and F helices.
HbS
However, under low oxygen concentration, HbS polymerizes and forms fibrous precipitates because the deoxy form of hemoglobin exposes a hydrophobic patch on the protein between the E and F helices.
– Another variant due to a variation in the β-chain gene
Hemoglobin C (α2βC2)
Hb C or HbC is an abnormal hemoglobin in which substitution of a (?) residue with a (?) residue at the 6th position of the β-globin chain has occurred (E6K substitution).
glutamic acid
lysine
This variant causes a mild chronic hemolytic anemia.
Hemoglobin C (α2βC2)
E6V substitution
Hemoglobin S (α2βS2)
E6K substitution
Hemoglobin C (α2βC2)
E26K substitution
Hemoglobin E (α2βE2)
– is an abnormal hemoglobin with a single point mutation in the β chain.
Hemoglobin E (α2βE2)
At position 26 there is a change in the amino acid, from glutamic acid to lysine (E26K).
Hemoglobin E (α2βE2)
is very common among people of Southeast Asian including Northeast Indian, East Asian descent
Hemoglobin E (α2βE2)
This variant causes a mild chronic hemolytic anemia.
Hemoglobin E (α2βE2)
– A heterozygous form causing sickle cell trait with one adult gene and one sickle cell disease gene.
Hemoglobin AS
– A compound heterozygous form with one sickle gene and another encoding Hemoglobin C.
Hemoglobin SC
These individuals have a mild hemolytic anemia and moderate enlargement of the spleen.
Hemoglobin SC
Persons with Hb SC disease may develop the same (?) complications as seen in sickle cell anemia, but most cases are less severe.
vaso-occlusive (blood vessel-blocking)
Patients with sickle cell trait (?) are protected from malaria.
(heterozygote for the sickle cell gene, Hb AS)
- the process of measuring the concentration of hemoglobin in blood.
Hemoglobinometry
- Determination of the concentration of hemoglobin in blood may be done using any of the following principles:
a. Methods based on the development of color when blood is mixed with a reagent.
b. A method based on specific gravity of the blood.
c. Measurement of oxygen combining capacity.
d. Indirect measurement using iron content.
e. By converting hemoglobin into one of several compounds and comparing the resulting compound with a known standard either visually or photoelectrically.
f. Electrical impedance or light absorption using automated hematology analyzers.
Laboratory determination of hemoglobin concentration
- Gravimetric Method (Specific gravity method or Copper Sulfate method)
- Gasometric method (Oxygen capacity method)
- Chemical method
- Colorimetric method
- Hemoglobin estimation using Automated Hematology Analyzers
- Hemoglobin Electrophoresis
– based on the estimation of specific gravity of blood, assuming that the patient has normal protein levels.
Gravimetric Method (Specific gravity method or Copper Sulfate method)
Specific gravity of copper sulfate of 1.053 corresponds to an Hb level of
12.5 g/dL
A drop of blood, allowed to fall into a copper sulfate solution of specific gravity 1.053, becomes encased in a +?), which prevents dispersion of fluid for 15 seconds
sac of copper proteinate
a. If the drop of blood falls in a few seconds,
it has a greater specific gravity than the solution
b. If the drop of blood rises in a few second,
it has a lower specific gravity than the solution
c. If the drop of blood remains suspended for about 15 seconds and then falls,
more or less it has the same specific gravity as the solution
This method is used by blood banks as a screening test for blood donors.
Gravimetric Method (Specific gravity method or Copper Sulfate method)
In most cases, this method is capable of estimating Hb within ~0.5 g/dL, which is comparable to a coefficient of variation (CV) of 2%
Gravimetric Method (Specific gravity method or Copper Sulfate method)
Based on the amount of oxygen in a given sample of blood, considering that hemoglobin will combine with and liberate a fixed quantity of oxygen.
Gasometric method (Oxygen capacity method)
(?)
The blood is hemolyzed with (?) and the oxygen is collected and measured in a (?)
Gasometric method (Oxygen capacity method)
saponin
Van Slyke apparatus
The oxygen combining capacity of blood is
1.34 ml O2 per gram of hemoglobin.
The volume of oxygen is corrected for
temperature and pressure
the hemoglobin concentration is determined with the use of the following formula:
An indirect measure of hemoglobin based on the amount of iron for a given sample of blood.
Chemical method
Based on the molecular structure, the iron content of hemoglobin is
0.347%
Thus, 1 gram or 1000 mg of Hb contains (?) of iron.
3.47 mg
The concentration of hemoglobin in blood is calculated by
dividing the iron content (mg/dl) by 3.47
(?)
Iron is detached from the hemoglobin by treating the blood with (?) in the presence of (?).
Wong’s method
concentrated sulfuric acid
potassium persulfate
(?)
The protein is precipitated with (?) and filtered out.
Wong’s method
tungstic acid
The iron content of the filtrate is determined in a colorimeter and the Hb value is calculated with the following formula:
The color of fresh blood is compared with a series of colored standards representing known quantities of hemoglobin.
Visual colorimetric method: Direct matching method
- In this method drop of blood is placed on filter paper and the color is matched with standard (Fig. 3-5[A]).
▪ Tallquist method
- In this method small glass chamber is filled with whole blood by capillary action. Then the glass chamber is illuminated by battery bulb. Color of the blood is matched with standard after seeing through eye piece.
▪ Dare’s method
- In this method the color of diluted oxyhemoglobin is matched visually.
▪ Spencer’s method
This method is less accurate than Sahli’s method. It is more difficult for the human eye to accurately grade and match small differences in red color than brown color of acid hematin.
▪ Spencer’s method
- This technique of estimating hemoglobin is based on comparing the color of a drop of blood absorbed on a particular type of chromatography paper against a printed scale of color corresponding to different levels of hemoglobin ranging from 4 to 14 g/dl.
▪ WHO hemoglobin color scale method
(?)
Blood is mixed with (?). This hemolyzes the red cells and converts the hemoglobin to a brownish yellow solution of acid hematin.
Acid hematin method
0.1 N HCl
is then compared with a colored glass standard (Comparator Block)
acid hematin
The procedures employed in the following are based on the principle of Acid Hematin method:
▪ Sahli – Hellige method
▪ Haiden – Hausser method
▪ Sahli – Adams method
▪ Osgood – Haskin method
▪ Haldane method
▪ Newcomer method
▪ Sahli –
Hellige method
Adams method
▪ Haiden –
Hausser method
▪ Osgood –
Haskin method
(?)
Blood is mixed with (?).
The solution is then boiled.
The hemoglobin is then converted to a blue–green solution of (?).
The color of the solution is then compared with a known standard or in a colorimeter
Alkali hematin method
0.1 N NaOH
alkaline hematin
Alkali hematin method will not accurately measure the hemoglobin of an infant, because infant’s blood contains
alkali resistant fetal hemoglobin (HbF)
The principle of Alkali – hematin method is used in the following:
▪ Standard method using Gibson and Harrison’s standard solution
▪ Clegg and King method
Principle:
Blood is mixed with either 0.1% sodium carbonate or 0.007 N Ammonium hydroxide solution. This converts the Hb to oxyhemoglobin. The depth of the resulting color is then measured in a photometer with a green filter (540 nm) and 0.007 N ammonium hydroxide as a blank
Oxyhemoglobin method
Principle:
Blood is diluted with Drabkin’s solution which contains ferricyanide and Cyanide. The potassium ferricyanide oxidizes hemoglobin to hemiglobin and potassium cyanide provides cyanide ions to form hemiglobincyanide, which has a broad absorption maximum at a wavelength of 540 nm. The absorbance of the solution is measured in a Photometer or spectrophotometer at 540 nm and compared with that of a standard HiCN solution.
Cyanmethemoglobin method (MHbCN method) or Hemiglobincyanide (HiCN) method
The concentration of hemoglobin in MHbCN method is computed using the following formula:
Hb determination is done by HiCN or the oxy-hemoglobin method. In the former, the blood specimen is diluted with a reagent containing ferricyanide and cyanide, which converts Hb to HiCN. The absorbance of the HiCN at 540 nm wavelength is then used for quantitation. In the latter, the blood specimen is diluted with an aqueous solution tetrasodium salt of ethylenediaminetetraacetic acid (EDTA) and mixed with air to convert Hb to oxyhemoglobin. The absorbance of oxyhemoglobin at 540 nm is then measured. A typical analyzer working on venous blood has a CV of ≤1.2% for Hb measurement.
Hemoglobin estimation using Automated Hematology Analyzers
These analyzers have become increasingly sophisticated in the last few decades with the incorporation of noncyanide methods. Hb determination is done using sodium lauryl sulfate (SLS), a surfactant that dissolves lipoproteins of the cell membrane of the red blood cells to release Hb and converts it into SLS-Hb. Concentration of SLS-Hb is measured as light absorbance and is calculated by comparison with the absorbance of the diluent measured before the sample is added
Hemoglobin estimation using Automated Hematology Analyzers
– Test for abnormal hemoglobins. This is generally considered the best method for separating and identifying hemoglobinopathies.
Hemoglobin Electrophoresis
One protocol for hemoglobin electrophoresis involves the use of two systems:
Cellulose acetate and agarose medium
Citrate agar
Initial electrophoresis is performed in alkaline buffers.
Cellulose acetate and agarose medium
are the major support media used because they yield rapid separation of HbA, F, S and C and many other mutants with minimal preparation time.
Cellulose acetate and agarose medium
because of the electrophoretic similarity of many structurally different hemoglobins, the evaluation must be supplemented by a procedure that measures some other property
Cellulose acetate and agarose medium
A simple procedure which confirms the identification of both HbS and HbC, as well as HbA, HbF and many other mutants
Citrate agar electrophoresis
This method is based on the complex interactions of the hemoglobin with the electrophoretic buffer (acid pH) and the agar support.
Citrate agar electrophoresis
Because some hemoglobins have the same charge and, therefore, the same electrophoretic mobility patterns, hemoglobins that exhibit an (?) may be subjected to electrophoresis at an acid pH for definitive separation.
abnormal electrophoretic pattern at an alkaline pH
In an acid pH some hemoglobins assume a negative charge and migrate toward the
anode
while others are positively charged and migrate toward the
cathode (negative pole)
Hb S migrates with Hb D and Hb G on (?) but separates from Hb D and Hb G on (?).
alkaline electrophoresis
acid electrophoresis
Similarly, Hb C migrates with (?) on alkaline electrophoresis but separates on acid electrophoresis.
Hb E and Hb O
The relative amount of hemoglobin is not proportional to the size of the band
in sickle cell trait (Hb AS), the bands may appear equal, but the amount of HbA
exceeds that of HbS
The main function of hemoglobin is to
transport oxygen.
since oxygen is (?), it has to depend on hemoglobin found in red blood cells for its transport to the different organs and tissues of the human body
non-water soluble
hemoglobin increases (?() in blood by about a hundredfold. this means that without hemoglobin, in order to provide sufficient oxygen to the tissues, blood would have to make a complete circuit through the body in less than a second, instead of the minute that it actually takes.
o2 solubility
That would take a mighty powerful heart, which in normal circumstances cannot be maintained by the human heart leading to increased (?) that may result to heart failure.
cardiac output
During oxygenation, each of the (?) in a hemoglobin molecule can reversibly bind one oxygen molecule.
four heme iron atoms
Approximately (?) of oxygen is bound by each gram of hemoglobin.
1.34 mL
Let’s follow the path of oxygen from the lungs to the peripheral tissues. Oxygen diffuses from the (?) of the lungs, little sacs at the end of the finely divided air passageways in the lung into the (?) of the bloodstream and then into the (?), where it binds to hemoglobin.
alveoli
capillaries
red blood cells
The concentration of oxygen is relatively high in the alveoli, about (?) which means that Hb is virtually 100% saturated in the lungs and all four heme molecules have an O2 molecule bound to them.
100 mmHg
The reference interval for arterial oxygen saturation is
96% to 100%
The affinity of hemoglobin for oxygen relates to the (?)
partial pressure of oxygen (PO2)
often defined in terms of the amount of oxygen needed to saturate 50% of hemoglobin, called the
P50 value
The relationship is described by the (?), which plots the percent oxygen saturation of hemoglobin versus the PO2 (Figure 3-8).
oxygen dissociation curve of hemoglobin
The curve is (?), which indicates low hemoglobin affinity for oxygen at low oxygen tension and high affinity for oxygen at high oxygen tension.
sigmoidal
among hemoglobin subunits contributes to the shape of the curve.
Cooperation
Hemoglobin that is completely (?) has little affinity for oxygen.
deoxygenated
The secret to hemoglobin’s success as an oxygen delivery molecule is the fact that it has (?) that communicate to each other.
four subunits
Evidence for this is provided by hemoglobin’s (?) in oxygen binding.
“cooperativity”
In other words, the binding of one O2 molecule affects the binding of others, as we can see by the following:
• In order to achieve 25% saturation (an average of 1 O2 molecule per hemoglobin), the amount of O2 needs to be about (?).
• In order to achieve 50% saturation (an average of 2 O2 molecules per hemoglobin), the amount of O2 needs to be about (?).
18 mm Hg
27 mm Hg
Therefore, it is easier to bind the than
second molecule of O2
Nobel Prize winners for Chemistry for their studies of the structures of hemoglobin and myoglobin.
Max Perutz and John Kendrew
Using (?), hemoglobin was found to have two different forms or shapes.
X-ray diffraction
The (?) is dependent on the presence or absence of oxygen.
conformation or shape
The experiments revealed that (?) has a relatively low attraction for oxygen, but when one molecule of oxygen binds to a heme group, the structure changes to the oxygenated form, which has a greater attraction for oxygen.
Therefore, the second molecule of O2 binds more easily, and the third, and fourth even more easily.
deoxyhemoglobin
The oxygen affinity of (?) is many times greater than that of (?).
oxy-hemoglobin
deoxy-hemoglobin
illustrates the relationship between oxygen saturation of hemoglobin and the partial pressure of oxygen.
oxygen dissociation curve
In the Normal (N) hemoglobin-oxygen dissociation curve, P50 is the partial pressure of oxygen (O2) needed for
50% O2 saturation of hemoglobin
In the Left-shifted (L) curve with reduced P50, it can be caused by decreases in (?) (e.g., multiple transfusion of stored blood), (?) (raised pH), (?) (PCO2), and/or (?).
2,3-bisphosphoglycerate
H+ ions
partial pressure of carbon dioxide
body temperature
A left shifted curve is also seen with hemoglobin F and hemoglobin variants that have (?) and in alkalosis.
increased oxygen affinity
In the (?) with increased P50 can be caused by elevations in 2,3-BPG (e.g., in response to hypoxic conditions such as in high altitudes), H+ ions (lowered pH), PCO2, and/or temperature.
Right-shifted (R) curve
A right-shifted curve is also seen with (?) and in the presence of hemoglobin variants that have decreased oxygen affinity.
pulmonary insufficiency, congestive heart failure, sever anemia
Factors that affect Hemoglobin affinity for Oxygen
- Partial pressure of oxygen
patient with arterial and venous PO2 levels in the reference intervals (80 to 100 mm Hg arterial and 30 to 50 mm Hg venous):
higher percent oxygen saturation
higher affinity for oxygen than a patient for whom the curve is normal
Shift to the left
patient with arterial and venous PO2 levels in the reference intervals (80 to 100 mm Hg arterial and 30 to 50 mm Hg venous):
a lower oxygen affinity
shift to the right
explains the lower affinity of hemoglobin for oxygen due to increases in the partial pressure of carbon dioxide (CO2) which eventually decreases the blood pH
Bohr effect
Whenever the human body undergoes increased cellular respiration, such as in strenuous physical activities, there is also an increase in metabolic activity within the tissues involved resulting in the production of CO2 as a metabolic waste product.
To transport CO2 through the venous blood, it diffuses into the red blood cells combining with water to form carbonic acid (H2CO3). This reaction is facilitated by the enzyme, carbonic anhydrase. The carbonic acid will then dissociate to release H+ and bicarbonate (HCO3-). The increase in H+ due to this reaction decreases the blood pH as explained by the Bohr effect.
In addition to hydrogen ions and carbon dioxide, a key allosteric effector of hemoglobin is
2,3-Biphosphoglycerate (2,3-BPG)
a small molecule made in red blood cells
2,3-Biphosphoglycerate
affects oxygen-binding affinity by binding in a small central cavity of deoxygenated hemoglobin. This shifts the equilibrium towards deoxy-hemoglobin
2,3-BPG
The presence of acids leads to:
• ↑ H+
• ↓ pH
• ↑ O2
This promotes formation of the deoxy form of hemoglobin, shifting the oxygen dissociation curve to the right, promoting oxygen release to actively respiring tissues.
presence of acids
At high altitude, when oxygen in the atmosphere is scarce because the air is “thinner,”:
• ↑ 2,3- BPG
• ↑ CO2
• ↓ O2
(helping hemoglobin to release more of its bound oxygen = ↑ aerobic capacity)
• ↑ 2,3- BPG
It takes about 24 hours forits levels to rise, and over longer periods of time, the levels continue to increase as part of the acclimation effect.
2,3-BPG
2,3-BPG does not bind to fetal hemoglobin
• ↑ metabolic rates
• ↑ thermal energy
• ↑ average kinetic energy
• ↑ temperature
• ↓ affinity for oxygen
Giving the developing fetus better access to oxygen from the mother’s bloodstream:
• ↑ 2,3- BPG
This results in tighter binding of oxygen relative to maternal hemoglobin.
2,3-BPG does not bind to fetal hemoglobin
.
Hb decrease its affinity for oxygen to facilitate delivery to the tissues; shifts the oxygen dissociation curve to the right such as when tissues are actively engaged in physical activity, these tissues would require and eventually receive more O2.
• ↑ temperature
Carbon dioxide
↑ cellular respiration
↑ metabolic activity
↑ CO2
↓ O2
↑ H+ ions
↑ 2,3-BPG
In response to higher temperature, the Hb decrease its affinity for oxygen to facilitate delivery to the tissues. Thus, increased temperature in the blood shifts the oxygen dissociation curve to the right such as when tissues are actively engaged in physical activity, these tissues would require and eventually receive more O2.
In response to higher temperature, the Hb decrease its affinity for oxygen to facilitate delivery to the tissues. Thus, increased temperature in the blood shifts the oxygen dissociation curve to the right such as when tissues are actively engaged in physical activity, these tissues would require and eventually receive more O2.
