PROTEIN
ANALYSIS
OF BODY
FLUIDS
Protein Contant
• 50 - 55 % Carbon
• 6 - 8 % Hydrogen
• 20 - 23 % Oxygen
• 15 - 18 % Nitrogen
Kjeldahl Technique
(Analysis for Nitrogen Content)
Protein precipitates upon the addition •
of trichloroacetic acid (TCA)
protein nitrogen
titration by NaOH
pH < 7 , heat
ammonium ions
pH > 7
acid
NH3
100
protein =
* N = 6.25 * N
16
Turbidometric Methods
• Often used for a similar concenteration
range in CSF or urine
• protein precipitates upon the addition
of TCA, sulfosalicilic acid or acetic acid
• these techniques are not specific to
protein since other acid - insoluble
substances such as nucleic acid also
percipitate
Colorimetric Techniques
Highly specific for proteins and peptids
• Biuret Method
• Folin - Ciocalteu
(phosphotungastomolybdic acid)
• Lowry (Biuret + Folin)
• Bradford Method
(coomassie brilliant blue G - 250)
Biuret Method
NH2 - CO - NH - CO - NH2
• copper salts in alkaline solution
form a purple complex with
substances contain two or more
peptide bonds
++
• Biuret reagent is Cu in
alkaline solution
Biuret assay
• This assay is based on Cu2+ interaction with
protein
• In alkaline solution, the copper ions form
tetradentate complexes with opposite pairs of
peptide bonded nitrogens
• These complexes produce a blue color that can be
measured at 550 nm
• The reaction is dependent on in part on peptide
bonds and not solely on amino acid moieties.
Biuret Method
Cupric ions react with •
peptide bonds under
alkaline conditions
producing a violetpurplish color
(copper sulfate + K- •
Na-tartrate + NaOH)
Measure color in •
SPEC at 540 nm
O
O
H N
H
R
Cu
O
OH-
H N
2+
H N
H
R
O
H N
2+
O
N H
H
R
Cu
O
N H
Purple biuret complex
The drawbacks of Biuret assay
• Lacks sensitivity
• It requires a relatively large sample size
• Because large amounts of material are not
always available, the Lowery assay, which
uses the Folin reagent to increase sensitivity,
was developed.
Lowery method
• This assay is essentially a biuret reaction that incorporates the use of of
Folin-Ciocalteu reagent for enhanced color development
• Its ten times more sensitive than the biuret assay
• It is believed that the enhancement of the color reaction in the Lowery
procedure occurs when the tetradentate copper complexes transfer
electrons to the phospho-molybdic/phosphotungstic acid complex
(Mo+6/W+6, Folin phenol reagent)
• Reduction of the Folin phenol reagent yields a blue color read at 750
nm
• Range of sensitivity: 5-100 mg/ml
Lowery method
• Advantages:
– Reliable method for protein quantification
– Little variation among different proteins
• Disadvantages:
– Many interfering substances
•
•
•
•
Detergents
Carbohydrates
Glycerol
…..
– Slow reaction rate (time required: 40 min)
– Instability of certain reagents
• Alkaline copper reagents is unstable and requires daily preparation
• The assay is photosensitive
• Proteins irreversibly denatured
Coomassie dye binding method
• This method is known as the Bradford
method
• This assay is based on the immediate absorbance shift from 465
nm to 595 nm that occurs when Coomassie Brilliant Blue G-250
binds to proteins in an acidic solution
• The dye has been assumed to bind to protein via electrostatic
attraction of the dye’s sulfonic acid groups.
• The Coomassie blue has been shown to interact chiefly with
arginine residues, but weakly with histidine, lysine, tyrosine,
tryptophan and phenylalanine residues.
Advantages:
Rapid (10 min)
Sensitive ( 25-200 mg/ml)
Disadvantages:
Some variability in response between different
purified proteins
Proteins used for this assay are irreversibly
denatured
Bicinchoninic acid (BCA) method
• Proteins react with alkaline copper II to produce
copper I.
• BCA then reacts with copper I to form an intense
purple color at 562 nm
• The macromolecular structure of the protein, the
number of peptide bonds and the presence of four
amino acids (cysteine, cystine, tryptophan and
tyrosine) have been reported to be responsible for
color formation
Advantages:
Single reagent
End product is stable
Fewer interfering substances than Lowry assay
Sensitive
Standard assay: 10-1200 mg/ml
Microassay: 0.5-10 mg/ml
Disadvantages:
Slow reaction time (40 min)
Proteins irreversibly denatured
Comments
• Specimen Collection and Storage
– Test specimens must be
– Nonhemolyzed
– Cell-free
– Lipemic sera should not be assayed
– Test tubes must remain covered
• Dust and dirt particle contamination
– Storage conditions
– Use of outdated reagents
Techniques of Protein
Separation
• Electrophorasis
• Chemical Precipitation
Serum Electrophorasis on
cellulose acetate in pH=8.6
Cathod (-)
anode (+)
origin
globulin
albumin
Serum Electrophorasis on
cellulose acetate in pH=8.6
Cathod (-)
gamma
anode (+)
alpha2 albumin
beta alpha1
Serum Electrophorasis on
cellulose acetate in pH=8.6
Chemical Precipitation
• Sreum proteins have been devised
to resolve albumin and the
globulins
• with the addition of sodium sulfate,
ammonium sulfate or methanol the
globulins lend to precipitate,
leaving albumin in solution
CLINICAL SIGNIFICANCES
• Normal Range :
–serum : 6 - 8 g/dL
(albumin : 3.5 - 5 g/dL)
(globulin : 2.5 - 3.5 g/dL)
(Alb/ Glo : 1.3 - 2)
–urine : 150 - 200 mg/dL
–CSF : 15 - 45 mg/dL
Hyperproteinemia
• Dehydration
–diarrhea
–vomiting
• cirrhosis
• infection
• tissue necrosis
Hypoproteinemia
• Nephrotic syndrome (incrase
amount of protein [>2g/dL]
loss by urine)
• kwashiokor
• burn
• hemorrhage
proteinuria
• Orthostatic proteinuria
• Bence - Jones proteinuria