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)

pH < 7 , heat

protein nitrogen ammonium ions

pH > 7

acid

titration by NaOH 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 MethodNH2 - 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 violet-

purplish color•)copper sulfate + K-

Na-tartrate + NaOH(•Measure color in

SPEC at 540 nm

N

N

OH

RH

OH

Cu2+

OH-

ON

N

HH

H

R

O

N

N

OH

RH

OH

Cu2+

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 proteinsProteins 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 reagentEnd product is stableFewer interfering substances than Lowry assaySensitive Standard assay: 10-1200 mg/mlMicroassay: 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 (-) anode (+)

gamma 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