Protein Structure, Function, and Analysis

Posted on Nov 16, 2024 in Biology

Proteins

Formed by amino acids (aa) with an amino group (bases) and a carboxyl group (acid).

The essential aa are synthesized by the body.

AA differ in the structure of side chains (R groups).

Amphoteric character: behave as charged or uncharged particles depending on the pH.

Acid pH

Positive charge

Basic pH

Negative charge

Hybrid or zwitterion ion: dipole with zero charge.

PI (Isoelectric Point)

  • aa with zero net charge
  • aa > 2 units per PI: net negative charge
  • aa < 2 units per PI: net positive charge
  • Ethernet frames PROTEINS

Protein Structure

Primary

aa order or sequence.

The sequence of genetic information from DNA to mRNA transcriptase.

Secondary

Management regularly and periodically in space along a direction.

3 forms:

  • α-helix
  • Folded sheet
  • Random coil

Tertiary

Bending or folding back on themselves in the secondary structure.

Unique to each protein.

Gives the biological properties.

Quaternary

Binding of several polypeptide chains.

Protein Classification

Simple (Contain only aa)

Globular

Polypeptide chains, folded, spherical or globular.

  • Albumin
  • Globulins
  • Histone

Fibrous

Polypeptide chains arranged in parallel (long fibers or sheets).

  • Collagen
  • Keratin
  • Elastin

2 Types

  • Myosin (muscle)
  • Fibrinogen

Conjugate (Proteins with other chemicals)

Non-protein component: Apoprotein.

Non-protein component: Prosthetic group.

Types

  • Nucleoprotein (DNA/RNA)
  • Mucoproteins (HC > 4%)
  • Glycoprotein (HC < 4%)
  • Lipoproteins
  • Metalloproteins (metal)

Protein Function

  • Enzymes: biological catalysts
  • Structural: class and tissues
  • Defensive or protective: antibodies
  • Transport of molecules: Hb/lipoproteins/albumin
  • Hormones
  • Receptors: convey the message into the cell
  • Oncotic pressure: exert pressure that opposes the flow of water from the blood capillaries

Serum Protein (6.2-8.5 g/dL)

Plasma

90% water.

Contains fibrinogen (absent in serum).

Serum Protein

Synthesized in the liver.

Ig tp not in Hb.

Determination Methods

Kjeldahl

Detects total N content.

Not used.

Biuret

Determines total serum protein.

Reference method.

Automated.

Lowry

Sample treated with alkaline solution of Cu.

Ultraviolet Absorption

Absorbs light at 210 nm.

Manual or automatic.

Refractometry

Measures refractive index.

Manual method.

Protein Electrophoresis

Definition

Based on the motion of charged particles in an electric field.

  • Negative particles migrate to the anode (+).
  • Positive particles migrate to the cathode (-).

Types

  • Free or Mobile: uses a liquid medium.
  • Zonal or Support: performed in an inert medium, better resolution.

pH and Buffer

Proteins are amphoteric (+ or – according to pH).

Commonly used: barbital buffer at pH 8.6.

Media Types

  • Gel Polyacrylamide: depends on load and particle size.
  • Starch Gel: depends on size and burden.
  • Paper: to separate small molecules.
  • Cellulose Acetate: most used.
  • Agarose Gel: method of choice.

Steps

Electrophoretic separation.

Protein fractions revealed with dyes.

Quantify fractions.

Quantification Fractions

  • Elution: cut fractions, eluted with solvent, measure A.
  • Densitometry: quantifies dye with special photometers.

Electrophoretic Fractions

α1-Globulin

  • α1-Antitrypsin: inactivates trypsin and plasmin.
  • α1-Lipoprotein: transports cholesterol and fat-soluble vitamins.
  • α1-glycoprotein: acute phase reactant.
  • Prothrombin: coagulation factor II.
  • Globulin Thyroid: transports thyroid hormones.

α2-Globulin

  • α2-Macroglobulin: neutralizes trypsin, plasmin, and kallikrein.
  • α2-Lipoprotein: transports lipids.
  • Haptoglobin: fixes Hb.
  • Ceruloplasmin: fixes copper.
  • Erythropoietin: stimulates erythropoiesis.

β-Globulin

  • β-Lipoprotein: transports lipids and hormones.
  • Transferrin: transports Fe++.
  • C3 and C4: complement components.
  • Hemopexin: transports heme.

Gamma-Globulins

Immunoglobulin (Ig).

Protein C reactive.

Albumin

Fibrinogen

Appears in beta and gamma fractions (if sample is plasma).

Alteration Electrophoretic Patterns

Liver Disease

Decreased albumin, β-globulin, gamma-globulin.

Inflammatory

Acute

Increased α1-globulin, α2-globulin, decreased albumin.

Chronic

Increased gamma globulin, albumin normal or decreased, increased α1, α2, and β-globulins.

Nephrotic Syndrome

Decreased albumin, gamma-globulin normal or decreased, increased α2-globulin.

Monoclonal Gammopathy

Increased gamma-globulins.

Liver Cirrhosis

Normal or decreased α, increased gamma-globulin.

Plasma Lipoproteins

Insoluble in water.

Amphipathic molecules.

Are solutions.