Blood - Biochemical Aspects Functions

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Blood - Biochemical AspectsFunctions

• 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)

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• 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)

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• 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

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• 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 ...

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• Male versus female
• Hematocrit ( volume that is red cells)
• 40-50 in males
• 35-45 in females

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ProteinsSee Lehninger Chapter 3-6

• Proteins are polyamino acids
• Macromolecules - MW 5000 - several million
• Insulin - MW 6000
• Hemoglobin - MW 68 000

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Amino Acid Structure
Protein Structure
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• 20 common amino acids (AA)
• Classified based on the properties of the R groups

Acidic Glutamic Acid
Basic Lysine
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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 lt 7.4

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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?

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• 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 pIs less than 7.4

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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

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• 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

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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

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Zone Electrophoresis of Plasma Proteins

-
globulins
albumin
g
b
a1
a2
pI
6.0
5.6
5.1
4.7
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Protein Separation

• Size Exclusion Chromatography (SEC)
• Porous matrix (sephadex)

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• Affinity chromatography
• molecule attached to a column that specifically
  binds the protein of interest
• Coenzyme / enzyme
• Antigen / Antibody

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• SDS-PAGE (polyacrylamide gel electrophoresis)
• Separates by size
• Proteins are complexed with SDS to give the same
  charge density

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Two Dimensional Electrophoresis
Decreasing Mr
Decreasing pI
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Functions of Plasma Proteins

• Maintenance of
• Colloid osmotic pressure (p)
• pH
• electrolyte balance
• COP relates to blood volume

DP p
Protein soln
Water
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• 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?

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• By analogy with the ideal gas law

• In blood, which protein contributes most to p?
• Low molecular weight, high concentration

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• 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

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• Hypotonic - lower p than normal
• Hemolysis of RBC

Hb
H2O
Ghost Cells
Hypertonic - higher p than normal Hemolysis of RBC
Crenated Cells
Hypertonic
1.5 NaCl
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Functions of Plasma Proteins (contd)

• 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)

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Function and Properties of Selected Plasma
Proteins

• Consider three abundant plasma proteins
• Structure, function
• Coagulation, fibrinolysis, complement

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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
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• 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

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• 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

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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

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• Basic 4 chain structural unit
• MW 2x55000 2x27000 160000

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• 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

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• 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
  B-lymphocytes)

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• Synthesis
• In lymphocytes (T and B)
• Made in response to presence of antigen
  (foreign macromolecule, virus particle etc.)

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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)

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a
b
g
disulfide
Triple dumbell model (EM)
450 Å
90 Å
D
D
E
as, bs and gs are intertwined
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• Function
• Blood coagulation (clotting)

Plasmin is end product of fibrinolytic
system Clot needs to be removed Not needed
forever Could embolize to lungs, brain
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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

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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

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Scanning Electron Micrograph of Red Blood Cells
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• 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)

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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

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• O2 depleted Hb solution placed in contact with
  O2(g)
• Equilibrium reaction
• Fraction (s) of Hb converted to oxyhemoglobin

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• Described by empirical equation

K depends on ionic strength and pH of Hb
solution n generally given as 2.5 -2.6
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• Binding of O2 to 4 heme sites given by

Equilibrium constants for different reactions
different Binding of first O2 relatively low
affinity 2nd, 3rd and 4th - much higher
affinity Cooperative effect
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• Compare with binding curve for myoglobin

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• Myoglobin - oxygen reaction

At equilibrium
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Acid Effect - O2 Dissociation

• 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

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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)
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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

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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

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• 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)

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• 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

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• 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

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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

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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

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• 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

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• Function
• Initially arrest bleeding through formation of
  platelet plugs
• Stabilize platelet plugs by catalyzing
  coagulation reactions leading to formation of
  fibrin

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• 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

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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

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• 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

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Fibrinolysis

• Results in dissolution of fibrin clot

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