Protein Structure and Properties

Protein Structure

• Primary structure is the amino acid sequence within a peptide or protein
• Primary structure is held together by covalent, peptide bonds
• Secondary structure is composed of highly regular local sub-structures
• Two types of secondary structures are alpha-helices and beta-pleated sheets
• Tertiary structure involves the three-dimensional folding of a single protein molecule
• Alpha-helices and beta-sheets are folded into a compact structure in the tertiary structure
• Quaternary structure constitutes a larger assembly of protein molecules or polypeptide chains (subunits) to form multimers
• A dimer contains two subunits while a trimer contains three subunits

Protein Properties and Separation

• Size, charge solubility, and biological activity impact protein properties and separation
• Size separation methods:
  • Gel permeation
  • Molecular sieving
  • Starch gel
  • Polyacrylamide gel
• Charge separation methods:
  • Ion exchange
  • Electrophoresis
• Solubility separation method:
  • Reverse phase chromatography
  • Salting out procedures
• Biological activity separation methods:
  • Affinity chromatography
  • Immunoassay procedures
  • Immuno electrophoresis

Plasma Protein Distribution

• Albumin makes up 60% of plasma proteins
• Globulin represents 35% of plasma proteins
• Fibrinogen makes up 4% of plasma proteins
• Other plasma proteins account for 1%

Protein Functions

• Proteins are used for transport and storage
• Help with fluid (water and electrolyte) balance
• Proteins play a role in acid/base regulation
• Involved in acute phase response, antibodies, and the immune system
• Proteins are building blocks that repair tissue
• Enzymes
• Hormones
• Clotting factors

Laboratory Measurements

• Laboratories can measure total protein
• Albumin levels
• Immunoglobulins (IgG, IgA, IgM)
• Immunoglobulin IgE (allergy and parasites)
• SPE (serum protein electrophoresis)
• Protein and peptide hormones
• Enzymes, fibrinogen, and clotting factors are lab measured

Protein Methods

• Spectrophotometry is used to measure total protein and albumin
• Protein electrophoresis
• Immunoassay and ELISA can also be used to measure proteins, especially hormone levels

Protein Electrophoresis Introduction and Principles

• Electrophoresis is the migration of charged particles in a support medium by an electric field
• Proteins are zwitterionic, meaning they can be negatively or positively charged depending on the pH of the solution
• The distance a particle travels depends on its molecular weight and overall charge
• Iontophoresis involves the migration of ions
• Zone electrophoresis involves the migration of macromolecules

Electrophoresis Clinical Applications

• Proteins
• Immunoglobulins
• Hemoglobin
• Isoenzyme/enzyme
• Lipoprotein

Electrophoresis According to Charge

• Charged molecules placed in an electric field migrate toward a positive (anode) or negative (cathode) pole, depending on their charge

Principle of Seperation

• Separation is based on charge
• Separation is based on size

Components of Electrophoresis

• Driving force/electrical power is a main component
• Support medium
• Buffer
• Sample
• Detecting system

Support Mediums

• Agarose gel:
  • It's purified agar support matrix
  • Has no electroendosmosis
  • It can be stained and read in a densitometer after electrophoresis
  • Allows for long-term storage
  • Requires only a small sample size of ~2-10 µL
• Cellulose Acetate:
  • Support media is normally dry and brittle
  • Becomes pliable when soaked in electrolyte buffer
  • Support media can be stained and read in a densitometer after electrophoresis
  • Allows for long term storage
• Polyacrylamide Gel:
  • Gels can be layered
  • Different pore sizes provide good separation of molecules of different sizes
  • Has good resolution
  • Can detect 20 serum protein fractions rather than 5

Electrophoresis Procedure

• Serum is applied to the support media, and the protein dissolves in the buffer, giving them an electric charge
• Applying a specific current for a specific time
• Electrically charged molecules migrate along the supporting media as current flows
• Negatively charged protein molecules migrate toward the oppositely charged electrode
• The sample separates into bands where each contains molecules of similar mobility

Staining of the Supporting Medium

• Staining fixes the protein to the membrane by denaturing
• Fractions become visible
• Decolorization removes background color
• Each column's peak is a different band of molecules

Densitometer

• A densitometer scans a stained strip
• Reports a graphical representation of the bands
• As a light beam passes through each stained band, the percent transmission is recorded
• A graph represents concentration
• A decrease in % T means the concentration increase
• Increase in % T is graphed as low peak or no peak
• The determination of the area is how each protein fraction can be calculated

Factors Affecting Migration Rates

• Molecular weight, size, and shape
• Molecular charge in the buffer
• Net charge of particles
• Type of supporting medium
• Temperature
• Electrical voltage
• Migration time

Serum Protein Electrophoresis

• From top to bottom (closest to the + electrode):
  • Albumin
  • Alpha-1-Antitrypsin
  • Alpha-2-Macroglobulin
  • Haptoglobin
  • Transferrin
  • Beta-lipoprotein
  • Complement
  • Origin
  • IgG
  • IgA
  • IgM

Relative Percent of Protein Bands

• Albumin: 53-65%
• Alpha-1 globulin: 2-5%
• Alpha-2 globulin: 7-13%
• Beta globulin: 8-14%
• Gamma globulin: 12-20%

Paraproteins

• Malignant clone of plasma cells that produces identical antibodies
• Antibody shows as a sharp band on electrophoresis
• M-band, M-spike, paraprotein, and myeloma
• Immune paresis: suppression of immunoglobulin production through monoclonal proliferation
• MGUS (monoclonal gammopathy of undetermined significance) requires monitoring

Paraproteins - Bence Jones protein

• Bence Jones Protein (BJP) is free monoclonal light chains, kappa (k) or lambda (λ)
• Light chains are filtered at the glomerulus because they have low molecular weight (~22,000 daltons)
• The presence of light chains implies de-differentiation due to their synthesis in excess of heavy chains (G, A, M)

Interpretation of Electrophoresis Results

• Albumin is the fastest protein, and forming the largest band, as well as closest to the positive electrode are key features
• The next 4 bands are globulins: alpha 1, alpha 2, beta, and gamma globulins

Abnormal Findings

• Elevated Albumin:
  • Severe water loss (dehydration)
• Decreased Albumin:
  • Poor diet (malnutrition)
  • Severe burns
  • Liver or kidney disease
  • Rheumatoid arthritis
  • Cancer
• Elevated Alpha-1 globulin:
  • Rheumatoid arthritis
  • Pregnancy
  • Infection
  • Cancer
• Decreased Alpha-1 globulin:
  • Liver or kidney disease
• Elevated Alpha-2 globulin:
  • Rheumatoid arthritis
  • Cancer
  • Heart attack
  • Chronic infections such as tuberculosis
• Decreased Alpha-2 globulin:
  • Liver damage
  • Leukemia
  • Hemolytic anemia
  • Hypothyroidism
  • Wilson's disease
• Elevated Beta globulin:
  • Liver disease
• Decreased Beta globulin:
  • Kidney disease
• Elevated Gamma globulin:
  • Chronic infection
  • Autoimmune disease
  • Some types of leukemia
  • Multiple myeloma
  • Liver disease
• Decreased Gamma globulin:
  • Kidney disease
  • Problems of the immune system