Unit 7 Proteins
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Reference range for serum or plasma total protein:
6.0 - 8.2 g/dL
Reference range for serum or plasma albumin:
3.5 - 5.2 g/dL
Reference range for serum or plasma albumin/globulin (A/G) ratios:
1.1 - 1.8
Reference range for a 24-hour urine excretion of protein as well as for random urine:
- 50 - 150 mg/day
- <10 mg/dL for random urine
Reference value for CSF protein and glucose:
- protein: 15 - 40 mg/dL
- glucose: 40 - 70 mg/dL or 2/3 of blood glucose
Describe the albumin test method(s):
- Bromcresol Green method (dye binding)
- Most common.
Dye binds selectively to Albumin → complex is green color
Bromcresol Purple is similar.
What is the test method for serum protein?
- Biuret method
- Won’t react with anything smaller than tri-peptides.
Isn’t overly sensitive.
Linear Range doesn’t go below 1.0 or 2.0 g/dL.
Reagents: Copper Sulfate (cupric form) in NaOH, KI (keeps in cupric form), and gobs of NaK-Tartrate (to maintain suspension of proteins)
Cupric ion interacts with two adjacent amide bonds (which is why there must be at least three peptides).
The number of amides available is the limiting factor, so the degree of the reaction indicates amount of bonds.
Which three methods are suitable for CSF and Urine Protein assays?
- TCA (Trichloroacetic Acid) method
- -Very caustic (pH around 0).
- -Used by some methods
- -A 13.5% solution is used.
- Sulfosalicylic acid (SSA) method
- -Less caustic (pH around 3.0).
- -Used by most methods.
- -A 10% solution is used.
- Coomassie Blue (dye binding)
- -Comes in blue-yellow color and turns blue when protein binds it (albumin and globulins).
- -Our Most Sensitive Method, measuring smallest amounts especially in urine. Linear range reaches down to 3 – 5 mg/dL.
What are the major proteins in the Albumin fraction of Cellulose Acetate Electrophoresis (and a couple albumin facts)?
- Transport molecule for:
- -Free Fatty Acids (FFA)
- -Many Hormones
Main protein effecting osmotic pressure (oncotic pressure).
Decreased in Hepatic Disease and Nephrosis.
What are the major proteins in the Alpha-1 Globulin fraction of Cellulose Acetate Electrophoresis?
– a protease inhibitor and one of the Acute Phase Proteins. 90% of this group.
- Alpha Lipoproteins (HDL)
- A1Acid Glycoprotein (AAG) – an Acute Phase Protein. (The major protein increased during
Some other Glycoproteins and Mucoproteins.
when present. (Embryonic protein. Cancer marker.)
What are the major proteins in the Alpha-2 Globulins fraction of Cellulose Acetate Electrophoresis?
- A2Macroglobulin – plasmin and protease inhibitor and an Acute Phase Protein.
- One of the largest proteins and the slowest to be lost in Nephrotic Syndrome.
– binds free hemoglobin and is an Acute Phase Protein. It is decrease in hemolytic conditions because binding Hgb makes it a different protein.
(VLDL – mainly TG)
– Carries copper and is an Acute Phase Protein. It is decreased in Wilson’s Disease.
What are the major proteins in the Beta Globulin fraction of Cellulose Acetate Electrophoresis?
– binds free heme groups.
– transports iron and is NOT an Acute Phase Protein. It decreased in inflammation.
C-Reactive Protein (CRP)
- B2-Microglobulin – tiny. Filtered at Glomerulus but reabsorbed by proximal
- tubule. When there are tubule problems,
- it is lost in urine.
– An Acute Phase Protein
Complement C-3 and C-4
– present in plasma but not in serum.
Beta Lipoproteins (LDL
– mainly cholesterol)
What are the major proteins in the Gamma Globulins fraction of Cellulose Acetate Electrophoresis?
- Immunoglobulins (antibodies):
- IgG – most abundant antibody
- IgM – first antibody to be produces.
- IgA – Usually a secretory antibody
- IgE – elevated in allergies or internal parasites.
- IgD - ?
In protein electrophoresis, what is the effect of ionic strength?
Low Ionic Strength: migration increased but you lose ability to isolate tight bands of proteins.
High Ionic Strength: tailing decreased but so is migration. Don’t get good separation. (Electroendosmosis increases as ionic strength increases.)
Ionic Strength must be optimized for specific sample type.
In protein Cellulose Acetate Electrophoresis, what is the effect on albumin and globulin when you over or under-apply sample?
Over-applied: scanned Albumin will be LOW and Globulin HIGH.
Under-applied: scanned Albumin will be HIGH and Globulin LOW.
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