Biochemsitry-2-3-HbM-en 1
Structure and function
of proteins
MYOGLOBIN, HEMOGLOBIN
(heme proteins)
Biochemsitry-2-3-HbM-en 2
Reversible binding of a Protein to a Ligand:
Oxygen-Binding Proteins
 MYOGLOBIN, HEMOGLOBIN
 Oxygen- poorly soluble in aqueous, diffusion
ineffective-few millimeters
 No AA bind O2
 Evolution- proteins –transport and store oxygen
 Hemoproteins (heme, reversible binding of oxygen)
 Heme- Fe
Biochemsitry-2-3-HbM-en 3
Myoglobin- single binding site for O2
 Transport O2 in muscle tissue
 Globins
 Protein ligand interaction can be described
quantitatively
Biochemsitry-2-3-HbM-en 4
Saturation curve of myoglobin
Biochemsitry-2-3-HbM-en 5
 Coordination of Heme in Hb/Mb
 Coordination bond of Fe2+ - histidine F8 proximal (F helix
 Partially to histidine E7 distal (in E helix).
Biochemsitry-2-3-HbM-en 6
Myoglobin x hemoglobin
Myoglobin – hyperbolic binding curve for oxygen
-oxygen-storage protein
-Hemoglobin- multiple subunits- O2 binding sites
-oxygen transport
Biochemsitry-2-3-HbM-en 7
Functions of hemoglobin and myoglobin
Biochemsitry-2-3-HbM-en 8
Hemoglobin
 Structure of Hemoglobin
 primary – AA
 secondary (a helixes A-H)
 Tertiary (space composition …)
 a helixes, hydrophobic inside,
hydrophilic outside,
hydrophobic pocket - heme
 Quartery (subunits)
Biochemsitry-2-3-HbM-en 9
 Coordination of Heme in Hb/Mb
 Coordination bond of Fe2+ - histidine F8 proximal (F helix
 Partially to histidine E7 distal (in E helix).
Biochemsitry-2-3-HbM-en 10
Biochemsitry-2-3-HbM-en 11
Quaternary structure, tetramer
Human
hemoglobin,
two alpha(red)
two beta(yellow)
subunits,
4 heme groups
4 subunits
- hydrophobic
- electrostatic interactions
Biochemsitry-2-3-HbM-en 12
0 20 40 60 80 100 120
100
80
60
40
20
0
PercentO2saturation
Partial pressure of oxygen (pO2, mmHg)
Muscle in
exercising
Relaxing
muscle Myoglobin
Hemoglobin
Artery
[O2]
Vein
[O2]
Environmental Oxygen Effects Binding Affinity
AdaptedfromGarrett&Grisham(1999)Biochemistry(2e)p.480
SIGMOID S -SHAPE
HYPERBOLIC
Biochemsitry-2-3-HbM-en 13
DEOXYHEMOGLOBIN OXYHEMOGLOBIN
CONFORMATION OF T
(TENSE, LOW AFFINITY)
CONFORMATION OF R
(RELAX, LOW AFFINITY)
LUNG/BLOOD
TISSUE/BLOOD
Biochemsitry-2-3-HbM-en 14
􀂙 Dioxygen binds cooperatively to hemoglobin!
The binding of O2 to hemoglobin enhances the binding of additional
O2 to the same hemoglobin (take advantage of high concentrations
of O2 in the lungs; sigmoid curve ). Binding of O2 to Myoglobin is
not cooperative; hyperbolic cure).
􀂙 Affinity of hemoglobin for O2 is pH dependent!
H+ and CO2 promote the release of bound dioxygen (for instance in
active tissues such as in muscles). Reciprocally, higher
concentrations of O2 promote the release of CO2 (e.g. in the lungs).
􀂙 Dioxygen affinity of the tetrameric hemoglobin is regulated
by 2,3-BiPhosphoGlycerate (lowered by the presence of BPG)!
Biochemsitry-2-3-HbM-en 15
Importance of 2,3-Bisphosphoglyceric acid (2,3Bisphosphoglycerate
or 2,3-BPG, also known as 2,3diphosphoglycerate
or 2,3-DPG)
-glycolytic intermediate 1,3-bisphosphoglyceric
acid (1,3-BPG).
-2,3-BPG is present in human red blood cells at
approximately 5 mmol/L.
-It binds with greater affinity to deoxygenated
hemoglobin (e.g. when the red cell is near
respiring tissue) than it does to oxygenated
hemoglobin (e.g., in the lungs) due to spatial
changes: 2,3-BPG fits in the deoxygenated
hemoglobin configuration), but not as well in the
oxygenated
-It interacts with deoxygenated hemoglobin beta
subunits by decreasing their affinity for oxygen, so
it allosterically promotes the release of the
remaining oxygen molecules bound to the
hemoglobin, thus enhancing the ability of RBCs to
release oxygen near tissues that need it most.
2,3-BPG is thus an allosteric effector.
Biochemsitry-2-3-HbM-en 16
(R)
relaxed state
(T)
tense state
The Transportation of Blood Oxygen
Hemoglobin
Lung
O2
Myoglobin
Muscle
Vein
ArteryWhen environmental [O2]
increases, Hb binds oxygen
efficiently
When environmental [O2]
decreases, Hb releases
oxygen to Mb
Any one subunit receives an oxygen molecule will increase the
oxygen-binding affinity of the others
Biochemsitry-2-3-HbM-en 17
0 20 40 60 80 100 120
100
80
60
40
20
0
PercentO2saturation
Partial pressure of oxygen (pO2, mmHg)
Muscle in
exercising
Relaxing
muscle Myoglobin
Hemoglobin
Artery
[O2]
Vein
[O2]
Environmental Oxygen Effects Binding Affinity
AdaptedfromGarrett&Grisham(1999)Biochemistry(2e)p.480
Biochemsitry-2-3-HbM-en 18
ALLOSTERISM
Biochemsitry-2-3-HbM-en 19
Hemoglobin transport H+ and CO2
 Products of cellular oxidation H+, CO2
 From the tissues to lung and kidneys
Biochemsitry-2-3-HbM-en 20
The Bohr Effect
 Most of the CO2 is transported as bicarbonate, which is formed within
 red blood cells by the action of carbonic anhydrase:
 CO2 + H2O HCO3− + H+
 􀂙 The major portion of the Bohr Effect is due to the fact that
 increasing p(CO2) causes a decreased red cell pH (acidosis).
 􀂙 A secondary part of the Bohr Effect is due to the fact that CO2
 reacts covalently with hemoglobin to form carbamino-hemoglobin
 which has a reduced O2 affinity.
 R—NH2 + CO2 R—NH—COO− + H+
 The bound carbamates form salt bridges that stabilize the T-form!
 (The Tense-form of hemoglobin possesses a lower O2 affinity).
a decrease in blood pH or an increase in blood CO2
concentration will result in hemoglobin proteins
releasing their loads of oxygen and a decrease in
carbon dioxide or increase in pH will result in
hemoglobin picking up more oxygen. Since carbon
dioxide reacts with water to form carbonic acid, an
increase in CO2 results in a decrease in blood pH.
Biochemsitry-2-3-HbM-en 21
Biochemsitry-2-3-HbM-en 22
Biochemsitry-2-3-HbM-en 23
Types of Hb
 Fetal Hb (HbF small increase affinity in
comparison to HbA)
 HbA1c – glycosylation
 HbA1c reference value:
 men 140-180 g/l ; 8,1-11,2 mmol/l
 women: 120-160 g/l ; 7,4-9,8 mmol/l
Biochemsitry-2-3-HbM-en 24
Hemoglobin- transport oxygen in bood
 Erythrocytes (red blood cells, 6-9um in diameter)
 Precursor stem cells- hemocystoblast – maturation- large amount of
hemoglobin, lose their nucleus, mitochondria, endoplasmatic
reticulum
 120 days survivor
Biochemsitry-2-3-HbM-en 25
Glycosylated hemoglobin is the form of hemoglobin
to which glucose is bound. The binding of glucose
to amino acids in the hemoglobin takes place
spontaneously (without the help of an enzyme) in
many proteins, and is not known to serve a useful
purpose. However, the binding to hemoglobin does
serve as a record for average blood glucose levels
over the lifetime of red cells, which is approximately
120 days.
The levels of glycosylated hemoglobin are therefore
measured in order to monitor the long-term control
of the chronic disease of type 2 diabetes mellitus
(T2DM). Poor control of T2DM results in high levels
of glycosylated hemoglobin in the red blood cells.
The normal reference range is approximately 4–
5.9 %. Though difficult to obtain, values less than
7% are recommended for people with T2DM.
Levels greater than 9% are associated with poor
control of the glycosylated hemoglobin, and levels
greater than 12% are associated with very poor
control.
Diabetics who keep their glycosylated hemoglobin
levels close to 7% have a much better chance of
avoiding the complications that may accompany
diabetes (than those whose levels are 8% or
higher).[61] In addition, increased glycosylation of
hemoglobin increases its affinity for oxygen,
therefore preventing its release at the tissue and
inducing a level of hypoxia in extreme cases.[62]
Biochemsitry-2-3-HbM-en 26
Role in disease
Hemoglobinopathies
 Genetic diseases (mutation in globin chain)
 Altered rate of Hb production (thalassemias)
Sickle cell hemoglobin HbS (mutation)
Biochemsitry-2-3-HbM-en 27
Hemoglobin je jen jedním z proteinů patřících do velké skupiny zvané hemové
proteiny (hemoproteiny).
Do této skupiny dále patří, např.:
myoglobin (vazba a depozice kyslíku ve svalech)
cytochromy (přenašeče elektronů v elektron-transportním řetězci mitochondrií)
katalázy a peroxidázy (rozklad a tvorba H2O2)
cytochrom P450 (hydroxylační systém/enzym)
Jejich společným znakem je to, že ve své molekule obsahují hem (cyklický
tetrapyrrol).
Hemoproteins
Biochemsitry-2-3-HbM-en 28
Hemoproteins protoporfirin
His- residue- heme coordination, oxygen on the other site
Fe2+--Fe3+, heme iron – higher affinity for CO (carbon monooxide),
- NO (nitric..) TOXIC to aerobic metabolism
-Blood: different color- oxygen rich- bright red arterial blood
-- dark purple oxygen depleted venous blood
Biochemsitry-2-3-HbM-en 29
Other hemoproteins
Catalysis
•Cytochrome P450s
•cytochrome c oxidase
•peroxidases
•Electron transfer/transport
•cyctochrome a
•Cytochrome b
•cytochrome c
The cytochrome P450 superfamily (officially abbreviated as CYP) is a large and diverse group of enzymes
that catalyze the oxidation of organic substances. The substrates of CYP enzymes include metabolic
intermediates such as lipids and steroidal hormones, as well as xenobiotic substances such as drugs and
other toxic chemicals. CYPs are the major enzymes involved in drug metabolism and bioactivation,
accounting for about 75% of the total number of different metabolic reactions.[1]
The most common reaction catalyzed by cytochromes P450 is a monooxygenase reaction, e.g., insertion of
one atom of oxygen into an organic substrate (RH) while the other oxygen atom is reduced to water:
RH + O2 + NADPH + H+ → ROH + H2O + NADP+
Cytochromes P450 (CYPs) belong to the superfamily of proteins containing a heme cofactor and, therefore,
are hemoproteins. CYPs use a variety of small and large molecules as substrates in enzymatic reactions.
Often, they form part of multi-component electron transfer chains, called P450-containing systems. The letter
in P450 represents the word pigment as these enzymes are red because of their heme group. The number
450 reflects wavelength of the absorption maximum of the enzyme when it is in the reduced state and
complexed with CO.
Biochemsitry-2-3-HbM-en 30
Peroxidases (EC number 1.11.1.x) are a large family of enzymes that typically
catalyze a reaction of the form:
ROOR' + electron donor (2 e-) + 2H+ → ROH + R'OH
For many of these enzymes the optimal substrate is hydrogen peroxide, but others
are more active with organic hydroperoxides such as lipid peroxides. Peroxidases
can contain a heme cofactor in their active sites, or alternately redox-active
cysteine or selenocysteine residues.