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14. Proteins


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List the 8 functions of proteins:
1. Structural
2. Hormonal
3. Catalytic (enzymes)
4. Storage/transport
5. Immunity
6. Receptors
7. Osmotic balance
8. Nutritional/maintenance
What characterizes primary structure?
-Linear amino acid sequence
-Disulfide bridges
What characterizes 2ndary structure?
Shape of chains - alpha helix or beta sheets
What characterizes tertiary structure?
The 3-D shape of entire chain; determines protein function.
What characterizes quaternary structure?
2 or more polypeptide chains held together noncovalently.
Which level of structure is disruptured by denaturation?
What are the components of a protein electrophoresis?
What are the negative APR's?
What are the normal APR's?
-C-reactive protein
which protein is associated with non-environmental emphysema?
Which protein is increased in lupus and rheumatoid arthritis?
Acid glycoprotein
What makes up the alpha-1 fraction of electrophoresis?
-Acid glycoprotein
What makes up the alpha-2 fraction?
What makes up the Beta-1 fraction?
What makes up the Beta-2 fraction?
-Complement (CRP)
What is Wilson's disease?
An inability to process copper correctly
What is an indicator of Wilson's disease?
Decreased Ceruloplasmin
Which APR is significantly elevated in infections?
C-reactive protein
Which protein is NOT lost in renal damage b/c of its large size?
What is the historical method for protein measurement?
What is the principle of the Kjeldahl method?
-Protein is digested w/ acid
-Nitrogen converted to NH4+
-Protein nitrogen expressed as total protein by 6.25 factor
What is the current precipitation method for protein called?
What is used for the turbidimetric method?
How is it done?
Sulfasalicylic acid - precipitates protein for scattered light measurement.
What is used to measure the light scatter in the turbidimetric method?
What is the principle of refractometry?
Proteins change refractive index of water in propertion to their concentration.
What are 2 drawbacks of refractometry?
-Only gives an estimate of protein concentration.
-Interferences = high BUN, hyperglycemic, lipemic, elevated albumin levels.
What is the principle of the Biuret method?
-Protein + Cu2+
-Alk pH via sodium tartrate
-Violet color forms by chelation of Cu and peptide bond
What wavelength is the biuret method read at?
540 nm
What are drawbacks of the biuret method?
-Hemolysis interferes
-Gross lipemia interferes
-Not useful for CSF/urine
What method is used for Albumin analysis?
-Dyebinding method
-What dye is used for Albumin?
-What needs to be kept in mind?
Brom-cresol Green
-Read at spcf time before the dye binds other proteins.
What protein method is useful for CSF and urine?
Immunochemical methods
How are immunochemical methods read for protein?
What charge is on the
Cathode = negatively charged
Anode = positively charged
So what migrates to the
Cations migrate to the cathode

Anions migrate to the anode.
What factors affect mobility in serum protein electrophoresis?
-Net protein charge
-Protein size, shape
-Electrical field
-Support used (gel, etc)
How does protein size affect electrophoresis?
-Larger proteins migrate slower
-Smaller proteins migrate faster
What is the definition of electrophoresis?
-Differential migration of charged particles in a liquid medium under an electrical influence causing them to seperate.
What type of medium is typically used for protein electrophoresis?
Cellulose acetate
What is the principle of isoelectric focusing?
-A pH gradient is set up on the solid medium
-Proteins migrate to their pI
-Allows specific seperation
How are urine samples prepped for electrophoresis?
Need to be concentrated
What things are looked for in urine protein electrophoresis?
-Bence jones proteins
Bence jones proteins in urine are an indicator of:
Multiple myeloma
What type of CSF has the most protein normally?
Ventric < Cistern < Lumbar
What are increased CSF proteins an indicator of?
Meningitis or BBB integrity damage
How is radial immunodiffusion used for protein measurement?
The area of diffusion in agar is specific for proteins, and allows quantitation of the concentration.
How is Ouchterlony analysis useful in protein analysis?
Allows for determining identity, partial identity, or non-identity of proteins.
How is Rocket electroimmunoassay analysed?
What type of protein is this typically done for?
By measuring the height of the precipitation the ANTIGEN concentration is measured.
What is "electro-endosmosis"?
A net flow of solvent to the cathode - an interferent
How does electroendosmosis interfere with protein electrophoresis?
It hinders solutes that want to go to the anode.
How does HEAT affect electrophoresis?
-Increases migration
-distorts patterns
-Denatures some proteins
-Increases ionic concentration of the buffer
What type of buffer enhances electrophoresis?
Buffers with a high ion cloud
What is a densitometer?
modified spec for quantitating seperated bands

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