Blood - Biochemical Aspects
Functions
• Respiratory
– Transport O2 from lungs to tissues
– Transport CO2 from tissues to lungs
• Nutrition
– Transport “food” from gut to tissues (cells)
• Excretory
– Transport waste from tissues to kidney
(urea, uric acid, water)
• Regulatory
– Water Content of Tissues
• Water exchanged through vessel walls to tissue
(interstitial fluid)
• Body Temperature
– Water- high heat capacity, thermal
conductivity, heat of vaporization
– Typical heat generation is 3000 kcal/day
• Protective
– Antibodies, antitoxins, white blood cells
(WBC)
• Blood composition
– 5-6 L in an adult
– 70 mL/kg of body weight
– Suspension of cells in a carrier fluid (plasma)
• Cells - 45% by volume
• Plasma - 55% by volume
• Cells
– Red cells (erythrocytes)
• 5x106/mL
– White cells (leukocytes)
• 7x103/mL
– Platelets (thrombocytes)
• 3x105/mL
• Plasma composition
– Water - 90% of plasma volume
– Proteins - 7% of plasma volume
– Inorganic - 1% of plasma volume
• Na+, K+, Mg2+, Ca2+, PO43-…
– Organic - 2% of plasma volume
• urea, fats, cholesterol, glucose ...
• Male versus female
– Hematocrit (% volume that is red
cells)
• 40-50% in males
• 35-45% in females
Proteins
See Lehninger Chapter 3-6
• Proteins are polyamino acids
• Macromolecules - MW 5000 - several
million
– Insulin - MW = 6000
– Hemoglobin - MW = 68 000
Amino Acid Structure
R
NH2CH
COOH
Protein Structure
O
O
~NHCHC-NHCHC~
R1
R2
Peptide bond
• 20 common amino acids (AA)
• Classified based on the properties
of the R groups
Acidic
Glutamic Acid
Basic
Lysine
Polar
Tyrosine
Apolar
Glycine
Amino Acids and Proteins
• Acidic and basic groups are charged at
blood / physiologic pH
• Proteins are polyelectrolytes
• pH of zero net charge (pI or isoelectric
point) depends on amino acid composition
of protein
• Blood proteins negative at pH 7.4
– more COO- than NH3+, pI < 7.4
pI
•
•
•
•
Protein has many negative charges
Requires H+ to neutralize
Therefore low pI
Consider a protein with pI = 4
– If pH increases above pI protein becomes?
– If pH decreases below pI protein becomes?
• Higher the pI the more +,- is protein?
• Need to go to a higher pH to
neutralize or compensate for +
charges
• Minimum solubility
occurs at pI since there is no
intermolecular repulsion
• At pH 7.4 (blood pH), all blood
proteins are negative and therefore
have pI’s less than 7.4
Protein Structure
• Four levels
– Primary structure: sequence of amino acids
• 20 amino acids in long chain molecules
• many possible combinations
– Secondary structure: arrangement of the
chains in space (conformation of chains)
 a-helix: coil shape (due to H bonding)
 b-sheet: stretched zig-zag peptide chain (H
bonding
• random coil: similar to synthetic polymers
– Tertiary structure: folding of chains into 3
dimensional shape due to H bonding, S-S
bonds and hydrophobic interactions
• Several different types of secondary structure
within the full three dimensional structure of a
large protein
– Quaternary structure: present in proteins
with several polypeptide chains, arrangement
and interelationship of the chains due to S-S
bridging
• Four levels result in well defined shape
and chemical structure essential for
function of protein
Plasma Proteins
• More than 200
• Most abundant
– Albumin - 4-5 g/100 mL
 g-glubulins - ~1 g/100 mL
– fibrinogen - 0.2-0.4g/100 mL
• Original classification by zone
electrophoresis at pH 8.6
• Separation by pI with several molecular
weight species within each group
Zone Electrophoresis of Plasma
Proteins
+
globulins
g
pI
6.0
b
a1 a2
5.6
5.1
albumin
4.7
Protein Separation
• Size Exclusion Chromatography (SEC)
– Porous matrix (sephadex)
• Affinity chromatography
– molecule attached to a
column that specifically
binds the protein of
interest
• Coenzyme / enzyme
• Antigen / Antibody
• SDS-PAGE (polyacrylamide gel
electrophoresis)
– Separates by size
– Proteins are complexed with SDS to
give the same charge density
Two Dimensional
Electrophoresis
Decreasing Mr
Decreasing pI
Functions of Plasma
Proteins
• Maintenance of:
– Colloid osmotic pressure (p)
– pH
– electrolyte balance
• COP relates to blood volume
DP = p
Protein
sol’n
Water
• If membrane present p important
• “Isotonic” - same osmotic pressure
• Human blood - 300 milliOsmoles /L
• Normal saline - 0.9% NaCl by weight
– 0.15 mol/L
– 0.30 mol/L of particles
• Calculate osmotic pressure from
concentration?
• By analogy with the ideal gas law
n
p  RT
V
 CRT (C in mol/L)
c

RT (c in g/L)
M
•In blood, which protein contributes most to p?
•Low molecular weight, high concentration
• Colloid - large particle that cannot
easily cross a membrane
– Stays in the compartment
– In blood pprotein = 20-30 mmHg
– Total ~ 5000 mmHg
• Protein stays in the blood as p is
maintained in the blood
• Water content is therefore
maintained
• Hypotonic - lower p than normal
– Hemolysis of RBC
Hb
H 2O
Ghost Cells
Hypertonic - higher p than normal
Hemolysis of RBC
Hypertonic
1.5% NaCl
Crenated Cells
H2O
Functions of Plasma Proteins
(cont’d)
• Transport of ions, fatty acids, steroids,
hormones etc.
– Albumin (fatty acids), ceruloplasmin (Cu2+),
transferrin (Fe), lipoproteins (LDL, HDL)
• Nutritional source of amino acids for
tissues
• Hemostasis (coagulation proteins)
• Prevention of thrombosis (anticoagulant
proteins)
• Defense against infection (antibodies,
complement proteins)
Function and Properties of
Selected Plasma Proteins
• Consider three abundant plasma
proteins
• Structure, function
• Coagulation, fibrinolysis,
complement
Albumin
• MW 66 000
• Single chain, 580 amino acids,
sequence is known
• Dimensions - Heart shaped molecule
• 50% a helix [He and Carter, Nature, 358
209 (1992)]
• Modeled as:
80 Å
30 Å
• Synthesis
– Mainly liver cells then exported
– Assembly time on ribosome ~ 1-2
min
– t0.5 in circulation - 19 days
– 14 g lost per day
– 0.4 mg synthesized per hour per g
of liver
– Need liver of approximately 1.5 kg
in weight to maintain
• Functions
– “Colloid” osmotic pressure of blood is
80% due to albumin
• relatively low molecular weight
• regulates water distribution
– Transport of fatty acids
• Liver to tissues, binding
– Source of amino acids for tissue cells
(pinocytosis)
• 60% albumin in tissue (interstitial) fluid
g-Globulins
• 20% of plasma proteins
• “g” refers to electrophoretic mobility
• Represents a group of proteins of
variable structure
– immunoglobulins
• Main functional task is immunochemical
– Antibodies - combine with specific antigens
• Basic 4 chain structural unit
– MW = 2x55000 +2x27000 = 160000
• Variable region varies with respect to
primary, secondary and tertiary
structures
• Basis of specificity of antigen binding
(106 average number)
• 5 classes of immunoglobulins
– IgG, IgA, IgM, IgD, IgE
– Different structures of constant regions
of heavy chains
– Some are polymers (multiples of 4 chain
unit - IgA - dimer - MW 350 000, IgM
- pentamer - MW 900 000
– See any immunology book for more details
• Functions
– Primary function is antigen binding
(immune response)
– Secondary function is complement
binding (after antigen)
– Each class has different functions
• IgE - allergic reactions (defence)
• IgA - secretory protein, high
concentration in external fluids (saliva,
tears)
• IgD - ? Involved in differentiation of B
lymphocytes (found on the surface of Blymphocytes)
• Synthesis
– In lymphocytes (T and B)
– Made in response to presence of
antigen (“foreign” macromolecule, virus
particle etc.)
Fibrinogen
• Coagulation
• Structure
– MW 340 000
– Sequence of amino acids is known
(3000)
– 4y, 3y structure
• 6 polypeptide chains, 2a (67,000), 2b
(56,000), 2g (47,000)
a
b
g
disulfide
Triple dumbell model (EM)
450 Å
90 Å
D
E
a’s, b’s and g’s are intertwined
D
• Function
– Blood coagulation (clotting)
Fibrinogen
Fibrin
Thrombin
Plasmin
Fibrin
Degradation (FDP)
Plasmin is end product of fibrinolytic system
Clot needs to be removed
Not needed forever
Could embolize to lungs, brain
Sickle Cell Anemia
• Occurs because of a minor variation in
one amino acid in the b chain of Hb
• Results in Hb that, when exposed to low
O2 concentrations precipitates into long
crystals
• Elongate cell
• Damage cell membrane
• Decrease in amount of RBC
Cellular Elements of Blood
• Red cells
– 40 - 50% of blood volume
– 5 x 106 cells /mL
– “bag” of hemoglobin
• non-nucleated
• no proliferation
• cell membrane in excess so that deformation does
not rupture
– Shape
• Biconcave disc
• 8 mm in diameter, 2.7 mm thick, volume ~ 90 mm3,
area ~ 160 mm2
Scanning Electron Micrograph of Red Blood Cells
• Why this shape?
– Area to volume ratio is high (maximal?)
– Facilitates diffusion of O2 and CO2
• minimal distance of contents from surface
• Originates in bone marrow (hematopoiesis)
– Molecular explanation based on the
properties of the proteins in the cell
membrane is found in Elgsaeter et al.
Science, 234, 1217 (1986)
Oxygen Binding of Hb
• Blood must carry 600 L of O2 from
lungs to tissues each day
– Very little carried in plasma since O2
only sparingly soluble
– Nearly all bound and transported by
Hb of RBC
– Possible for Hb to carry four O2
molecules, one on each a chain, one on
each b chain
• O2 depleted Hb solution placed in
contact with O2(g)
• Equilibrium reaction
• Fraction (s) of Hb converted to
oxyhemoglobin
• Described by empirical equation
s
K ( pO2 )
n
1  K ( pO2 )
n
K depends on ionic strength and pH of Hb solution
n generally given as 2.5 -2.6
• Binding of O2 to 4 heme sites given
by:
Hb  O2  HbO2
HbO2  O2  Hb (O2 ) 2
Hb (O2 ) 2  O2  Hb (O2 ) 3
Hb (O2 ) 3  O2  Hb (O2 ) 4
Equilibrium constants for different reactions different
Binding of first O2 relatively low affinity
2nd, 3rd and 4th - much higher affinity
Cooperative effect
• Compare with binding curve for myoglobin
• Myoglobin - oxygen reaction
k1
Mb  O2  MbO2
At equilibrium
k 1
k1CMbCO2  k 1CMbO2
s

CMbO2
CMbO2  CMb
k1
CMbCO2
k 1
k1
CMbCO2  CMb
k 1

KCO2
1  KCO2
Acid Effect - O2
Dissociation

HHb  O2  HbO2  H

• O2 binding causes release of H+
• pH decreases, [H+] increases then the
equilibrium moves to left
• % saturation decreases, more
dissociation for a given pO2
• Tissues are at a lower pH than the
lungs due to CO2 which facilitates
release of O2 to tissues
Hb versus Mb
• Hb carry O2 to tissues where it is
released
– Releases quickly in tissues where pO2
is lower
• Mb store O2 in the muscle, make
available to cells
– Releases very little in tissues
Reference: Science 255 54 (1992)
RBC - Reversible Shape
Changes
• Surfactants result in cells becoming more
spherical
• Mechanical stress - deformation in
capillaries to allow for passage of cells
• Disease eg. Sickle Cell Anemia
• Hemolysis - release of Hb from the cell
– Osmotic swelling
– Surface collisions with artificial organs
White Blood Cells
(Leukocytes)
• Total count - approximately 7000/mL
• Various types
–
–
–
–
–
–
Neutrophils 62%
Eosinophils 2.3%
Basophils 0.4%
Monocytes 5.3%
Lymphocytes 30%
Plasma cells (mainly in the lymph)
• Monocytes in tissue become macrophages
• Function
– Defense against foreign invaders
• bacteria
• viruses
• foreign materials (including biomaterials)
• Phagocytosis
– Neutrophils, macrophages
– Move to foreign particle by chemtaxis
• Chemicals induce migration
• Toxins, products of inflamed tissues, complement
reaction products, blot clotting products
– Response is extremely rapid (approx 1 h)
• Lymphocytes
– B cells - responsible for humoral
immunity
– T cells - responsible for cell mediated
immunity
• B cells responsible for production of
antibodies
– Receptor matches antigen
– Cells multiply
– Antibodies
• Abs are just immunoglobulins
discussed earlier
• T cells
– Cytotoxic T cells (Killer T cells)
• Bind to cytotoxic cells (eg infected by
virus)
• Swell
• Release toxins into cytoplasm
– Helper T cells
•
•
•
•
Most numerous
Activate B cells, killer T cells
Stimulate activity by secretion of IL2
Stimulate macrophages
– Suppressor T cells
• Regulate activities of other cell types
AIDS
• HIV - attacks many cell types
–
–
–
–
epithelial cells
macrophages
neurons
lymphocytes (helper T)
• Infected helper T cells when stimulated,
produces viral proteins which kill the cell
• Helper T cell population disappears
Platelets
•
•
•
•
•
•
Non-nucleated disk shaped cells
3-4 mm diameter
Volume 10 x 10-9 mm3
250 000 cells/mL
10 day circulation time
Surface contains membrane bound
receptors (GP Ib and IIb/IIIa)
– mediate surface adhesion reactions,
aggregation reactions
– interact with coagulation proteins
• Contain muscle proteins actin and
myosin which contract when platelet
is activated
• Also a granules, dense granules,
lysosomal granules
• Platelets activated by minimal
stimulation
– Become sticky
– Shape change
– Release of cell contents
• Stimulate other platelets
• Function
– Initially arrest bleeding through
formation of platelet plugs
– Stabilize platelet plugs by catalyzing
coagulation reactions leading to
formation of fibrin
• Platelet Adhesion
– Site of injury - exposure of
connective tissue elements (eg
collagen)
– Artificial surfaces through forming
thrombi (clots)
• Platelet Aggregation
– Caused by ADP, collagen, thrombin,
epinephrine, PAF, TXA2
• Release of cell contents
– Induced by ADP, collagen, thrombin,
TXA2 and epinephrine
Coagulation
• Maintenance of hemostasis (prevention of
blood loss)
• At least 12 plasma proteins interact in
series of reactions
• Cascade of reactions
• Inactive factors become enzymatically
active following surface contact,
proteolytic cleavage by other enzymes
• Amplification is rapid
• Reactions are localized
• Extrinsic system
– Blood comes in contact with
traumatized vascular wall or
extravascular tissues
• Intrinsic system
– Initiated by surface contact (often
negatively charged surface)
• Most reactions are Ca++ dependent
• Chelaters of Ca++ effective
anticoagulants
Fibrinolysis
• Results in dissolution of fibrin clot