Unit 7 Proteins

6.0 - 8.2 g/dL

3.5 - 5.2 g/dL

1.1 - 1.8

• 50 - 150 mg/day
• <10 mg/dL for random urine

• protein: 15 - 40 mg/dL
• glucose: 40 - 70 mg/dL or 2/3 of blood glucose

• Bromcresol Green method (dye binding)
• Most common.

Dye binds selectively to Albumin → complex is green color

Bromcresol Purple is similar.

• 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.

• 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.

• Transport molecule for:
• -Free Fatty Acids (FFA)
• -Bilirubin
• -Drugs
• -Many Hormones

Main protein effecting osmotic pressure (oncotic pressure).

Decreased in Hepatic Disease and Nephrosis.

A1Anti-Trypsin (AAT) – 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
• inflammation.)

Some other Glycoproteins and Mucoproteins.

A1Fetoprotein when present.  (Embryonic protein.  Cancer marker.)

• A2Macroglobulin – plasmin and protease inhibitor and an Acute Phase Protein.
• One of the largest proteins and the slowest to be lost in Nephrotic Syndrome.

Haptoglobin – binds free hemoglobin and is an Acute Phase Protein. It is decrease in hemolytic conditions because binding Hgb makes it a different protein.

Pre-Beta Lipoproteins (VLDL – mainly TG)

Ceruloplasmin – Carries copper and is an Acute Phase Protein. It is decreased in Wilson’s Disease.

Hemopexin – binds free heme groups.

Transferrin – transports iron and is NOT an Acute Phase Protein.  It decreased in inflammation.

• B2-Microglobulin – tiny. Filtered at Glomerulus but reabsorbed by proximal
• tubule.  When there are tubule problems,
• it is lost in urine.

C-Reactive Protein (CRP) – An Acute Phase Protein

Complement C-3 and C-4

Fibrinogen – present in plasma but not in serum.

Beta Lipoproteins (LDL – mainly cholesterol)

• 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 - ?

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.

Over-applied: scanned Albumin will be LOW and Globulin HIGH.

Under-applied: scanned Albumin will be HIGH and Globulin LOW.