CHAPTER 10

Hemoglobin Metabolism
HEMOGLOBIN STRUCTURE

Hb
first protein whose structure was described using
x-ray crystallography
conjugated globular
4 heme groups and 2 heterogeneous pairs of
polypeptide chains
34 g/dL in RBC, 64,000 D
transport oxygen from lungs to tissues
modulates vascular dilation by transporting NO
transports CO2 from tissues to lungs

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HEME STRUCTURE

protoporphyrin IX + Fe
2+
= ferroprotoporphyrin
1 heme molecule = 1 oxygen molecule
double bonds = red

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GLOBIN STRUCTURE

4 globin chains in 1 heme
consist of 2 identical pairs of unlike polypeptide
chains, 141 to 146 amino acid each
each chain is divided into 8 helices (AH) and 7
nonhelical segments

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COMPLETE HB MOLECULE

A. Primary structure
amino acid sequence of polypeptide chains
B. Secondary structure
chain arrangements in helices and nonhelices
C. Tertiary structure
arrangement of helices in pretzel-like
configuration
D. Quaternary structure
tetrameric molecule
spherical, 4 heme groups attached to 4
polypeptide chains and may carry 4 molecules
of oxygen
2 -globin and 2 non- globin chains


Each heme group is suspended between helices E
and F
Heme iron is positioned b/w 2 histidine radicals
proximal bond F8 and distal histidine residue in E7

Distal histidine swings in and out to permit passage of
oxygen

Globin chain AAs in the cleft are hydrophobic.
Outside AAs are hydrophilic (w/c makes it water soluble)

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HEMOGLOBIN BIOSYNTHESIS

HEME BIOSYNTHESIS

occurs in mitochondria and cytoplasm of RBC bone
marrow precursors (pronormoblast to reticulocyte)
loss of mitochondria and TCA = no more Hb synthesis

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GLOBIN BIOSYNTHESIS

chromosome 16: and
chromosome 11: , , ,
one copy of each globin gene per chromatid for a
total of two genes per person w/ the exception of
and (2 copies each per chromatid, total of4 genes
per person)
pronormoblast to reticulocytes
arise via transcription of genetic code to mRNA and
translation of mRNA to globin polypeptide chain
in pronormoblasts: <
translation: <
when synthesized, chains are released from the
ribosomes in the cytoplasm

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HEMOGLOBIN ASSEMBLY

HbA = 22
HbA2 = 2 2
HbF = 2 2 in F cells

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HEMOGLOBIN ONTOGENY

chromosome 16:
chromosome 11: , ,
first 3 months of embryonic development: and
at birth: HbF is predominant

HbA1C
most glycated Hb
glucose attaches to N-terminal valine of the chain
increased in diabetes mellitus

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HEMOGLOBIN PRODUCTION REGULATION

HEME REGULATION

key rate limiting step: Gly + succinyl CoA ALA

heme inhibits
a. transcription of ALAS gene
b. ALA dehydrase
c. porphobilinogen
deaminase/hydroxymethylbilane synthase
increased heme demand = increased ALAS
synthesis

Ferrochelatase
heme synthase
plays a regulatory role in heme biosynthesis
inhibited by heme negative feedback or
substrate inhibition by protoporphyrin IX

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GLOBIN REGULATION

Globin production is regulated by the rate at which
the DNA is transcribed to mRNA
globin=mRNAs

Hemin
Fe
3+
oxidation product of heme
impt in controlling the rate of globin synthesis in
intact reticulocytes and various cell-free systems
w/o it, inhibitor of globin synthesis accumulates

Excess components of Hb (unpaired chains,
protoporphyrin, iron) reduce RBC survival.

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HEMOGLOBIN REGULATION

Hb synthesis is stimulated by tissue hypoxia.
During hypoxia, kidney produces increased EPO
w/c stimulates Hb and RBC production.

Reference intervals:
Men: 14-18g/dL (140-180 g/L)
Women 12-15 g/dL (120-150g/L)
Newborns: 16.5-21.5 g/dL (165
to 215 g/L)

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HEMOGLOBIN FUNCTION

Hb readily binds oxygen molecules in the lungs (
O2 affinity)
and efficiently unload oxygen to the tissues (O2
affinity)
1.34 mL of oxygen per each gram of Hb
Affinity of Hb depends on partial pressure of oxygen
(PO2)

PO2
amount of oxygen needed to saturate 50% Hb
P50 value

Oxygen dissociation curve
percent oxygen saturation of Hb versus PO2

Hb that is completely deoxygenated has little affinity for
oxygen.. per oxygen molecule bound, avidity increases
and HB then becomes fully oxygenated.


Bohr effect
shifts in curve

27mm Hg = 50% oxygen saturation of Hb
shift to the left: <27 mmHg
shift to the right: >27 mmHg

96% to 100%
reference interval for arterial oxygen saturation

shift to the left: higher % O2 saturation and affinity
shift to the right: lower affinity

Shift to the right
lower affinity
CO2, H
+
, Cl
-
(strengthen salt bridges)
2,3-BPG (R T)
body temperature
decreased pH
presence of abnormal Hbs w/ low affinity for oxygen
high fever
acidosis
hypoxia (high altitude)
pulmonary insufficiency
congestive heart failure
severe anemia
cardiac righ-to-left shunt

Shift to the left
body tempearature
depleted 2,3-BPG
alkalosis
presence of Hb variants

Hb: sigmoidal curve
Mb: hyperbolic curve


Myoglobin
greater oxygen affinity than Hb
17000D monomeric
releases oxygen only at very low partial pressures
not as effective as Hb in releasing oxygen
released when there is damage to the muscle
like MI, trauma, severe muscle injury
(rhabdomyolysis)
excreted through the kidney
levels may become elevated in renal failure

HbF
P50 of 19-21 mmHg
left shift

Hemoglobin also transport CO2
CO2 + H2O H2CO3 (carbonic anhydrase)
H2CO3 H
+
+ HCO3
-
(Cl shift, lungs)

Carbaminohemoglobin
lower affinity for oxygen than does Hb in the
absence of CO2

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VARIANT HEMOGLOBINS

METHEMOGLOBIN

ferric iron
continuously being formed by spontaneous
oxidation but fails to accumulate
brownish to bluish
shift to the left
if >30% hypoxia and cyanosis
increased: nitrites or metHb reductase activity,
HbM disease
assayed by spectral absorption analysis (CO-
oximeter)
peak: 620-640 nm at a pH of 7.1
methemoglobinemia may be treated by removal
of offending substance or administration of
ascorbic acid or methylene blue

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SULFHEMOGLOBIN

formed by irreversible oxidation of Hb by
sulfonamides, phenacetin, acetanilide,
phenazopyridine,
created in vitro by addition of hydrogen sulfide to
Hb
greenish pigment
if elevated cyanosis
peak: 620nm
does not shift when cyanide is added

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CARBOXYHEMOGLOBIN

combination of carbon monoxide w heme iron
some are produced endogenously
541 nm
cherry-red
treatment: hyperbaric oxygen

Carbon monoxide
silent killer
odorless and colorless
affinity is 240x, release is 10,000x slower
exposure may be coincidental, accidental or
intentional (suicidal)
toxic effects: headaches, dizziness, coma,
convulsions

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HEMOGLOBIN MEASUREMENT

CyanmetHb method
1. Lysing agent frees Hb
2. Free Hb combines w/ potassium ferricyanide
contained in cyanmetHb reagent
3. Ferrous to ferric metHb
4. MetHb + potassium cyanide cyanmetHb
5. CyanmetHb measured at 540nm


Sodium lauryl sulfate (SLS)
convert Hb to SLS-metHb
does not generate toxic wastes

Hb electrophoresis
separate different types of Hb