Download QUANTITATIVE DETERMINATION OF BLOOD PLASMA PROTEINS

Medical chemistry and Biochemistry
2nd year
QUANTITATIVE DETERMINATION OF BLOOD PLASMA PROTEINS BY
ELECTROPHORESIS
Practical training follows the lecture “Plasma proteins”.
Blood plasma proteins are responsible for many physiological functions. Changes in the
spectrum of blood proteins do reflect patient’s health status and its electrophoretical
analysis helps with the diagnosis. Frequently observed changes are peak elevations or
depression in the electrophoreogram. For instance the elevation of α1 and α2 fractions is
associated with the progress and presence of the acute inflammation, and elevation of γglobulins reveals the presence of chronic inflammation.
The decrease of total blood protein concentration (hypoproteinemia) is frequently
associated with the malnutrition. The most abundant protein, albumin, helps in
maintaining of the oncotic pressure. Decrease of the total albumin induces osmotic
liquid transfer to the interstitial space, and, subsequently induces the formation of
edema. Hyperproteinemia is more commonly connected to organism dehydration.
Presence of, so called, paraprotein in electrophoreogram strongly suggests malignant
tumour (Kahler’s syndrome). Specific procedure – immunoelectrophoresis – enables
estimation and identification of the required/exact pathological protein. It is necessary
to keep in mind that some quantitative changes in protein spectrum can be the
consequence of protein loss due to the kidney disease.
The aim of this practical training is to learn methods of protein estimation and explain
the interpretation of electrophoreogram.
1. SAMPLE PREPARATION
All tasks will require serum. Following lecturer’s instruction and under the laboratory
technician’s surveillance take out the capillary blood into the microtube. Incubate 10
min. at room temperature and then centrifuge at 4000 RPM for 5 min. Transfer
separated blood serum into clean microtube. In order to be able to carry out following
experiments, you should prepare at least 10 µL of serum (for electrophoresis).
2. PROTEIN ESTIMATION
Total serum proteins concentration varies in between 62-82 g/L.
2.1 Total protein – biuret reaction
In the presence of peptides, a Cu2+ ion forms violet-coloured coordination complexes in
an alkaline solution (Fig.1), which can be estimated by spectrophotometry at 545 nm.
Medical chemistry and Biochemistry
2nd year
Fig.1
Sensitivity of this method is 0.1-10 mg of protein.
Into 3 test-tubes pipette the solution according to the following table:
Biuret reagent [mL]
Standard [mL]
Sample [mL]
Dist.water [mL]
Blank
1
0.02
Sample
1
0.02
-
Standard
1
0.02
-
Tab.1
Stir well and incubate for 10 min, at laboratory temperature. Measure the absorbance of
the sample and standard against blank solution.
Total protein concentration in standard is………….g/L.
Total protein concentration in sample is…………….g/L.
Calculation:
2.2 Total protein - Lowry method
Folin-Ciocaulteau’s phenol reagent reacts with aromatic sidechains of the aromatic
amino acids (tyrosine, tryptophan) in proteins and amplifies the intensity of biuret test.
Resulting compound is of blue colour. Response is dependent on amino acid
composition of the protein. Estimation is carried out spectrophotometrically at 720 nm.
Sensitivity is 10-200 μg of protein.
Into 3 test-tubes pipette the solution according to the following table:
Working solution is prepared by addition of 9.8 mL of solution I (20 g Na2CO3 + 0.5 g of
sodium potassium tartrate in 1 L of 0.1 mol/L NaOH) and 0.2 mL of solution II (0.1%
CuSO4).
Medical chemistry and Biochemistry
2nd year
Working solution [mL]
Standard [mL]
Sample [mL]
Dist.water [mL]
Blank
2
0.02
Sample
2
0.02
-
Standard
2
0.02
-
Tab.2
Mix well and incubate for 15 min, at laboratory temperature.
Then add:
Folin-Ciocaulteau’s reagent dilute in ratio 1+1 with distilled water before use.
F-C reagent [mL]
Blank
0.1
Sample
0.1
Standard
0.1
Tab.3
Test-tubes shake well and incubate for 30 min, at laboratory temperature. Measure the
absorbance of the sample and standard against blank solution.
Total protein concentration in standard is ……………..g/L.
Total protein concentration is ……………g/L.
Calculation:
3. ELECTROPHORESIS OF THE SERUM PROTEINS
Protein electrophoresis is a well established technique routinely used in clinical
laboratories for screening of serum and some other fluids for protein abnormalities. It is
based on the principles of zone electrophoresis performed on a suitable support
medium. Agarose has been developed into a versatile and effective support medium.
In diagnostic applications, serum proteins separate into six major fractions, primarily
according to their charge at a given pH (pH 8.6): albumin, alpha-1 globulins, alpha-2
globulins, beta-1 globulins, beta-2 globulins and gamma globulins.
3.1 Agarose gel – ready to use
3.2 Sample preparation and application


Apply 10 µL of serum sample into the applicator wells. Load the applicator
within 40 sec.
Snap off the applicator teeth's protection frame. The applicator so comes into
contact with the surface of the gel. After 1 min, turn the switch to rise up the
applicator, remove the applicator and discard.
Medical chemistry and Biochemistry
2nd year
3.3 Electrophoresis



Pour the electrophoresis chamber (pH 8.6).
Put the gel into an appropriate electrophoresis chamber, according to the
polarity indicated on the gel, the lower side of the gel on the cathodic side.
Connect the power supply, adjust voltage to 90 V and run 22 min.
3.4 Finalization
 After finishing the electrophoresis switch off the power supply and remove
the lid.
 Carefully remove the gel and then dry the gel at 80 °C air for about 10 min.
3.5 Staining and destaining
 Immerse the dried and cooled gel in the staining solution (Amidoblack) for 5
minutes.
 Destain in destaining solution until the background is completely colorless
and clear.
3.6 Scanning
 Scan using a densitometer.
4. ELECTROPHOREOGRAM INTERPRETATION
Agarose electrophoresis can detect up to 6 fractions:
Fraction
Plasma Proteins
%
Albumin
albumin
55.8-66.1
α1-globulins α1-protease inhibitor
α1-acid glycoprotein (orosomucoid)
2.9-4.9
α1-fetoprotein
α1-lipoprotein
α1-microglobulin
α2-globulins α2-macroglobulin
haptoglobin
7.1-11.8
ceruloplasmin
ferritin
β1-globulins transferrin
4.7-7.2
hemopexin
β2-globulins β2-microglobulin
3.2-6.5
β-lipoprotein
complement components C3 a C4
γ-globulins Immunoglobulins (G,D,M,A,E)
11.1-18.8
Tab.4
Medical chemistry and Biochemistry
2nd year
Fibrinogen
Fig.2: Electrophoresis and densitometric record of serum proteins1
Sometimes, the fractions β1 and β2 are not separated and only one fraction, β, is
detected.
Pathologic states are demonstrated on electrophoresis by elevation, decrease or
disappearance of some fraction. Even, new fractions with atypical mobility can be
detected.
Always the proportion of the fraction has to be related to the total serum protein
concentration.
Discussion:
1. What would you detect in the resulting electrophoreogram of serum proteins of
patient with acute attack rheumatoid arthritis? (remark.: RA is chronic
inflammatory disease)
2. Why is blood serum used? What would be the difference if non-clogging blood
will is used?
3. Try to interpret the pathological electrophoreograms.
4. The pathological findings would be accompanied by increased or decrease ESR
(Erythrocytes Sedimentation Rate)?
5. Are there physiological changes of electrophoreogram? If yes, describe?
Conclusion:
1
Racek et. al: Klinická biochemie, Galén, 2006