Flashcards in Quiz 4: Proteins Deck (33)
• Amino group is removed by deamination or transamination
• Resulting ketoacid enters metabolic pathways with carbohydrates and fats.
• The ammonium ion produced during protein breakdown is converted to urea in the Liver and excreted through urine
• Nitrogen balance
• Catabolism occurs in Liver
Basic Makeup of Proteins
• Complexes of carbon and nitrogen
• Covalently bonded amino acids - COOH of amino acid links to NH2 of another amino acid, with removal of H20 & forms a peptide bond.
N-terminal: end with free amino group
C-terminal: end with free carboxyl group
Contain 16% Nitrogen, separating it from lipids and carbohydrates
Levels of Protein Structure
• Primary Sequence is linear sequence (arrangement) of amino acids from end to end (from N-terminal to C-terminal) - form polypeptide chain
• Secondary Structure is the winding of the polypeptide chain, how the chain folds onto itself into sheets and helixes (alpha helix, beta sheets)
- Maintained by hydrogen bonds between the NH & COOH groups
• Tertiary Structure is the twisted folds that form the 3D conformational shape due to interaction between atoms in chain (globular)
• Quarternary Structure the arrangement of 2 or more different polypeptide chains interacting to give a unique 3D spatial arrangement.
Causes of Denaturation
Can be by heat, hydrolysis by strong acids or alkali, enzymatic actions, exposure to urea, exposure to UV.
Highest concen. of plasma proteins
Soluble in water, globular, includes many enzymes.
Regulates osmotic pressure, transport proteins due to easy binding with blood components
Increase and decrease in levels follow hypo/hyperproteinemia causes
Soluble in dilute aqueous solutions with added ions (NaCl)
Proteins linked to lipids
Proteins linked to carbohydrates (antibodies, cell surface receptors)
The protein can be either positive or negatively charged, depending on pH of the environment.
When a protein’s number of negative groups = number of positive groups, no net charge
• If protein is in pH > pI, protein is negative
• If protein is in pH < pI, protein is positive
• Soluble proteins have charge on their surface dependent on the number/type of amino acids and pH. If uncharged, protein interactions lead to protein precipitation (lowest solubility is at the protein’s pI)
• A hydrophilic protein prefers to interact with water due to it’s charge (more soluble)
+ Proteins in aqueous solution swell and enclose water when reconstituting:
- lyophilized serum (control vials), it is important to mix gently to allow complete swelling
“Colloidal emulsoids”: protein molecule enveloped by water.
+ Albumin remains in solution at higher concentration because it holds onto water.
• Simple: Proteins with peptide chains that upon hydrolysis yield amino acids (Globular: Albumin, and Fibrous proteins: collagen & troponin)
• Conjugated: composed of a protein (apoprotein) plus a non-protein (lipid, carbo, etc) "prosthetic group" , grouped based on their components (Lipoproteins, glycoproteins, mucoproteins, nucleoproteins, metalloproteins)
• Production of energy (citric acid cycle)
• Distribution of water
• Buffers (amphoteric nature)
• Specific transporters of metabolic substances
• Glycoproteins (immunoglobulins and immunity)
• Receptors for hormones (hormonal message transmission)
• Structural function (collagen, bone, tendons, cartilage)
• Clotting factors in hemostasis
• Rare, inherited disorders of amino acid metabolism (>100)
• Phenylketonuria (PKU)
• Maple Syrup Urine Disease
• Isovaleric Acidemia
Total Protein Range and Testing
• Usual method is biuret photometric procedure.
• Additioanlly used: Refractometer, Electrophoresis, Dye binding
• Analysis is done on plasma, serum, urine, CSF, amniotic fluid, saliva, peritoneal fluid, pleural fluid etc.
• Total protein in other body fluids (CSF, urine etc) are much lower (mg/dL) and alternative methods to the biuret procedure are required.
Kjeldahl Protein Assay
• Was Gold standard method, now considered reference method as it is too cumbersome for routine testing
• Measurement of total protein by protein precipitation, followed by acid protein digestion.
- Nitrogen is converted to ammonium bisulfite, which is measured.
• Calculated by a conversion factor from protein nitrogen to ammonia to total protein. Nitrogen is determined; an average of 16% nitrogen mass is assumed to calculate protein concentration.
• Cupric ions (Cu+2) are complexed with the groups involved in the peptide bonds in alkaline solution.
• Colormetric measurement proportional to the number of peptide bonds present.
• Urine, CSF etc protein levels are ~ 0.01 g/dL.
Dye Binding Method
• Coomassie Brilliant Blue G250
• When protein binds to the dye the absorbance at 595 nm increases.
• Limited use for total protein in serum because not all proteins bind with the same equal dye binding (absorbance) characteristics.
• UV light (280 nm) is absorbed by phenyl rings in tyrosine, tryptophan and phenylalanine amino acids in the protein.
• Limited use with mixed proteins (serum) but good with pure protein solutions.
Total Protein Indications
• Protein elevated in inflamm., MI, infections, tumors etc.
• Diagnostic information determined by the albumin, globulin fractions and the A/G ratio.
• A/G ratio: Total Protein – Albumin = Globulin
• Abnormal results are followed by electrophoresis
Negative nitrogen balance
Caused by excess protein loss (urinary), GI Tract inflammation, Liver disorders, malnutrition, inherited immunodeficiencies, and extensive burns
Elevation of all protein fractions from dehydration
Measuring Urine and CSF Total Protein
Dye using Coomassie Brilliant Blue
Turbidimetric using Acid to precipitate the protein
Dye-binding with Bromocresol Green or HABA, which increases the maximum absorpance of the dye proportionally to the concen. of Albumin
Urine and Serum electrophoresis are done the same way, but urine must be concentrated first
Sample added to Cathode end of gel then electrophoresed
Separate in this order: Albumin, alpha-1, alpha-2, beta, gamma
Stains used include Amido Black, Ponceau S, and Coomassie Brilliant Blue
* If plasma is used instead of serum, a fibrinogen peak will appear between the beta and gamma regions
Liver produces most of the plasma proteins (Albumin, alpha-1, alpha-2 and beta)
RE produces gamma globulins
Causes of Decreased Albumin
Lowered synthesis in Liver
Alpha-1 antitrypsin: increased in acute phase and pregnancy, lowered in emphysema
- Acute phase reactant, inhibits trypsin-type enzymes that damage structural proteins
Alpha fetoprotein: High in Neural Tube Defects Low with fetuses with Downs Syndrome, Marker for Liver Cancer
- Normally only found in yolk sac and liver of fetus, measured during pregnancy as "multiples of the median - MoM"
Haptoglobin: High in acute phase/nephrotic syndrome, Lowered in Transfusion Reactions, hemolysis, and Liver Disease
Ceruloplasmin: Transports Copper, lowered in Wilson's disease
- 90% serum copper is bound to ceruloplasmin
Macroglobulin: Proteolytic enzyme inhibitor to thrombin, trypsin, and pepsin