Download Proteins

Proteins
 Contain carbon, hydrogen, oxygen, nitrogen, and sulfur
 Serve as structural components of animals
 Serve as control molecules (enzymes)
 Serve as transport and messenger molecules
 Basic building block is the amino acid
General characteristics
 Molecular size:
 Proteins are macromolecules.
 Structure:
 Proteins have four levels of structure:
 Primary.
 Secondary.
 Tertiary.
 Quaternary.
 Nitrogen content:
 The average N content is 16%.
 This characteristic is used for the measurement of total protein.
 Charge and isoelectric point (PI):
 Because of their a.a`composition, proteins can bear +ve and –ve charges (amphoteric
nature).
 The pH at which an a.a` or protein has no net charge is known as its isoelectric point.
This characteristic is used for separation and quantitation of proteins such as
electrophoresis.
 Solubility:
 Protein solutions are colloidal emulsoids because they are charged and because each
molecule of protein has an envelope of water around it.
Proteins compete for water in the presence of high conc. Of salt. By using a range of salt conc., a wide
array of specific proteins can be separated
 Immunogenicity:
 Because of their molecular mass, their content of tyrosin, and their specificity by
species, proteins can be effective antigens, human serum proteins bring forth the
formation of antibodies specific for each of the proteins present in the serum.
 Methods using antigen-antibody reactions are called immunoassays and nucleic acid
probe techniques.
 Synthesis:
 Most plasma proteins are synthesized in plasma cells.
General protein functions
 1. Enzymes:
 Have catalytic activity.
 Example: trypsin.
 2. Transport proteins:
 Example: hemoglobin, transports O2.
 3. Nutrient and storage proteins:
 Example: casien milk protein.
 4. Contractile and mobile proteins:
 These proteins give the ability to contract, to change shape or to move.
 Example: actin and myocin of skeletal muscle.
 5. Structural proteins:
 Example: Keratin found in hair.
 6. Defense proteins:
 Example: antibodies.
 7. Regulatory proteins:
 Example: hormones regulate cellular or physiological activity, like insulin.
Catabolism and nitrogen balance
 Protein catabolism and synthesis are equal, normally this turnover totals about 125-220 g of
protein/day, it varies widely for individual protein; Plasma proteins and intracellular proteins are
degraded rapidly (half-life hours-day) while collagen “for example” its half-life lives for years.
 This balance between catabolism and anabolism of protein is called Nitrogen Balance (NB).
 -ve NB: when catabolism > anabolism which occurs during tissue destruction, burns, and
high fever.
 +ve NB: when anabolism > catabolism, formed during growth, pregnancy and repair
process.
Protein classification
 1. Simple proteins:
 Contain peptide chains.
 Two types:
 Fibrous proteins, ex. collagen.
 Globular proteins, ex. albumin.
 2. Conjugated proteins:
 Consists of a.a` chains and non-a.a` molecules.
 The a.a` portion is called apoprotein, the non-a.a` portion is called prosthetic group.
Conjugated protein
Prosthetic group
Lipoprotein
Lipid, ex. cholesterol
Glycoprotein
Carbohydrates (<4% of molecule) ex. haptoglobulin
Mucoprotein
Carbohydrates (>4% of molecule) ex. mucin
Metalloprotein
Metal, ex. hemoglobin
Nucloprotein
DNA and RNA (chromatin)
Phosphoprotein
phosphate
Estimation and clinical significance of the individual plasma proteins
 The most important plasma proteins:
 Albumin.
 α1-Globulin (α1-antitrypsin)
 α2-Globulin (haptoglobulin)
 β-Globulin (transferrin, fibrinogen)
 -Globulin (immunoglobulin)
Albumin
 Molecular weight 66,300 Dalton.
 It has the highest conc. in the serum.
 Reference value: 35-55 g/l (adult).
 Function:
 Bind and transport bilirubin, steroids, and fatty acids.
 Regulation of osmotic pressure.
 Pathological abnormalities:
 Decreased conc. of serum albumin may be caused by:
 Malnutrition (low intake of a.a`).
 Liver disease (cirrhosis).
 Gastrointestinal loss (inflammation, disease of the intestinal mucosa).
 Renal disease.
α1-Antitrypsin
 It is the main α1-globulin.
 It is an acute phase reactant.
 Reference value 2-4 g/l (adult).
 Function:
 It neutralizes the action of trypsin-like enzyme (elastase) that cause hydrolytic damage
to structural protein (ex. elastin).
 Pathological abnormalities:
 Decreased conc. of serum α1-antitrypsin occur in case of hyaline membrane disease
(respiratory distress syndrome).
 Juvenile hepatic cirrhosis.
 Increased conc. of serum α1-antitrypsin occur in:
 Inflammatory reaction, pregnancy, and oral contraception..
 Methods for determination:
 Abnormal level of α1-antitrypsin is made by the lack of α1-globulin band on protein
electrophoresis.
Immunological methods are used for quantitation
Haptoglobulin
 It is an α2-glycoprotein.
 Synthesized in hepatocytes.
 Its level in the serum increases with age, with a marked increase in males.
 Function:
 Binds to haemoglobin by its α-chain.
 Haptoglobulin prevents the loss of haemoglobin into urine.
 Pathological abnormalities:
 Low levels of haptoglobulin indicates hemolysis or acute hepatocellular damage.
 High levels are seen in:
 Inflammation, Burns,
 Methods for determination:
 Gel filtration.
 Immunological methods are used for quantitation.
Transferrin
 It is one of the β-globulin.
 Function:
 Transport iron.
 Pathological abnormalities:
 Low levels of transferrin are seen in patients with hereditary transferrin deficiencies
(marked hypochromic anaemia).
 Methods for determination:
 Immunological methods.
Fibrinogen
 It is one of the β-globulin.
 It is one of the largest proteins in the plasma, its MWT 341,000 Dalton.
 Function:
 Precursor of fibrin clot.
 Pathological abnormalities:
 Low levels of fibrinogen reflect extensive coagulation (fibrinogen consumed).
 High levels of fibrinogen occur in case of inflammation.
 Methods for determination:
 Fibrin clot isolation, then assay by Biuret method.
Phosphate salt ppt method, then assay by Biuret method.
Immunoglobulins
 Five classes: M, A, G, D and E.
 They are antibodies that are synthesized in the plasma cells.
Immunogloulins
MWT
Referance value
Comments
IgG
150,000
8-12
Antibodies
IgA
180,000
0.7-3.12
Antibodies in secretion
IgM
900,000
0.5-2.8
Early response to
antibodies
IgD
170,000
0.005-0.2
Antibodies
IgE
190,000
6×104
Antibodies (allergy)
 Pathological abnormalities:
 Low levels of Igs are seen in immunodifficient disease.
 High levels of Igs occur in case of inflammation, chronic infection, and liver cirrohsis.
 Methods of determination:
 ELISA.
 Immunodiffusion
Proteins in other body fluids
 Urine protein:
 Normal level of total protein in urine is 12 mg/dl or 100 mg/24hrs.
 Proteinuria:
 Due to increase conc. of total protein in urine > 12 mg/dl.
 It is classified as resulting from either:
 Glomerular (loss of membrane integrity).
 Tubular dysfunction (no reabsorption).
Urine protein
Method of determination
Method
Principle
Comments
Turbidimetric
Proteins are ppt as fine particles,
turbidity is measured
spectophotomerically
Rapid, easy to use; unequal
sensitivity for individual
protein
Biuret
Proteins are ppt and re-dissolved in
alkaline solution, then reacted with Cu+2
the color produced is proportional to
conc.
Accurate
Folin-lowry
Oxidation of aromatic a.a`s by Folin
phenol reagent
Very sensitive
Dye-binding (commassie
blue, ponseaue S)
Protein bind to dye, cause shift in the
absorption
Limited linearity; unequal
sensitivity for individual
protein
Proteins in other body fluids
 CSF proteins:
 Reference value of total protein in CSF 15-45 mg/dl.
 Clinical significance:
 Increased total CSF proteins may be found in:
 Bacterial, viral, and fungal meningitis.
 Multiple sclerosis.
 Neoplasm (tumor plasma).
 Low CSF proteins values are found in case of hypothyrodism.
 Methods for determination:
 Sulfosalicylic acid method.
 UV spectrophotometry.