Serum protein electrophoresis

INTRODUCTION

Protein electrophoresis is a well established technique routinely used in clinical laboratories for screening of
serum and some other fluids for protein abnormalities. The separation of proteins, primarily accrording to their
charge at given pH, is performed on a suitable support medium, e.g. agarose gel.

The protein components of plasma or serum constitute almost all the mass of the serum solutes (approximately
80 g/L). The proteins perform a number of functions: carrier proteins, antibodies, enzyme inhibitors, clotting factors,
and so on. Serum is the specimen of choice. Plasma can be also used; however, an extra band produced by
fibrinogen will be observed (approximately 3 g/L) - a protein that is absent from serum. Serum proteins are
commonly classified according to the electrophoretic separation into 6 classes because the agarose gel
electrophoresis separates all the proteins to the six fractions:

1. albumin (ALB)
2. alpha-1-globulins (α1)
3. alpha-2-globulins (α2)
4. beta-1-globulins (β1)
5. beta-2-globulins (β2)
6. gama-globulins (γ)

The total serum protein concentrations or the proportions of the individual protein fractions change during a
variety of diseases: the electrophoretic analysis is commonly used in diagnostics of diseases. Urine and
cerebrospinal fluid can also be analyzed but only after previous increase of their protein concentration made by
ultracentrifugation, or other special techniques. The fluids must be concentrated because they contain less proteins
than blood serum does.

USED METHOD

Electrophoretic analysis is based on the separation of proteins in an electric field according to their relative
electrophoretic mobility. When an electric field is applied to a medium containing charged particles, the negatively
charged particles migrate toward the positive electrode (anode), while the positively charged particles migrate
toward the negative electrode (cathode).

Supporting media fall into two main classes: 1) supports which allow separation of molecules solely on the basis
of net molecular charge (e.g. cellulose acetate, agarose gel); 2) supporting media exerting a sieving effect on the
compounds being separated (e.g. polyacrylamide gel): the gel can be considered as a porous medium in which the
pore size is of the same order as the protein molecules. The result is that a molecular sieving effect is observed in
addition to the electrostatic separation and hence the resolution power of these gels is much greater: there are
about 25 or more serum proteins bands observed on polyacrylamide gel in comparison with six bands observed on
agarose gel. The many bands seen with polyacrylamide support medium present a complex picture that is hard to
interpret and appears to help little in routine clinical laboratory diagnosis. Except for specialized purposes,
polyacrylamide cannot be recommended for routine serum protein electrophoresis.

The separation is commonly carried out at pH 8.6, which is higher than the pI (= isoelectric point pH) values of the
major serum proteins. Proteins are amphoteric molecules: they are either uncharged or negatively or positively
charged particles, depending on the pH of a buffer used for analysis. The buffer helps to maintain a constant pH,
and it ensures that each protein will maintain a constant charge throughout the course of the separation. If the
buffer of pH 8.6 (commonly barbital or Tris-barbital buffer) is used the most of proteins are negatively charged and
move toward the anode at a rate dependent on their net charge. Major peaks (bands) observed in order of their
migration are those of albumin, alpha-1 globulins (α1), alpha-2 globulins (α2), beta-1 globulins (β1), beta-2
globulins (β2) and gamma globulins (γ). Albumin has the highest mobility from all of the fractions. Some of
these peaks represent many of different proteins that have a similar migration rate at pH 8.6. However, certain
proteins predominate in each peak and variation in their relative amounts is characteristic of certain diseases.

In practice, it is desirable to have rapid separation of proteins to preserve sharpness of the bands. The longer the
electrophoresis is allowed to proceed, the greater the radial diffusion and the broader the bands. One can decrease
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the time for a given separation by either decreasing the length of the support or increasing the voltage. With an
increase in voltage, there will be a corresponding current increase, and heating may become a limiting factor
because of protein denaturation. One way of reducing heating is by lowering of the ionic strength of the buffer. Use
of constant voltage is a standard practice in serum protein electrophoresis: separation is achieved within about
30 minutes at about 90 to 100 volts.

The separated proteins are fixed (by acid / alcohol mix or by hot air) in the support medium. Fixation causes
precipitation of the separated proteins. Staining the proteins (e.g. by amidoblack solution; the medium should be
dried in an oven before the staining to evaporate the previous solvent) and subsequent scanning is the most
popular method of quantitation of individual protein fractions. Densitometer is used for scanning of separated
proteins in a gel, see Fig. 1. Scanning the pattern gives quantitative information about well-resolved protein
fractions only. Observation with an experienced eye is helpful in the detection of minor fractions and unsatisfactory
separations.

Fig. 1: Separated protein fractions of blood serum are represented on the left side. Densitometer graph is
illustrated on the right side.
Note: Beta-fraction can be separated into beta-1 and beta-2 globulins in agarose gel. Six peaks will be in
densitometer graph.

Major proteins found in each fraction

The list below summarizes principal proteins of each of the fractions, and proteins found between the neighbouring
fractions - in interzones (the proteins are of lower concentration than the proteins of individual fractions).

Albumin is synthesized in the liver. Its serum concentration is 35 - 55 g/L and half-life 20 days. Albumin is very
important binding and transport protein of numerous substances (fatty acids, hormones, bilirubin, drugs, metals).
This protein is also included in controlling the fluid balance between intravascular and extravascular compartments
of the body (maintenance of oncotic pressure). Albumin is a one of buffer systems in blood plasma.
- increased: dehydration
- decreased: liver disease, chronic infection, kidney disease, malnutrition, monoclonal gammopathies

Albumin /alpha-1 interzone

Alpha 1-lipoproteins (= high density lipoproteins, HDL) participate in transport of cholesterol and fat-soluble
vitamins.
- increased: hyperlipidemia
- decreased: liver disease
Alpha 1-fetoprotein is an oncofetal antigen synthesized early in fetal development.
- increased: liver carcinoma
Alpha 1-acid glycoprotein (orosomucoid) is a glycoprotein synthesized in the liver.
- increased: in response to a variety of acute and chronic inflammatory stimuly
- decreased: if the liver proteosynthesis is damaged

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Alpha-1 globulins
Alpha 1-antitrypsin (= alpha 1-protease inhibitor, API) is an inhibitor of proteolytic enzymes (e.g. trypsin) derived
from leucocytes, bacteria, or endogenous production. This substance belong among „acute phase reactants
(proteins)“.
- increased: acute inflammatory reactions
- decreased: hypoproteinemia, inherited conditions

Alpha-1 /alpha-2 interzone

Alpha 1-antichymotrypsin is an inhibitor of proteolytic enzymes.
- increased: acute inflammatory reactions

Alpha-2 globulins
Alpha 2-macroglobulin is also inhibitor of proteolytic enzymes. Its concentration is dependent on age (higher in
childhood).
- increased: nephrotic syndrome
- decreased: rheumatoid arthritis, myeloma
Haptoglobins are a group of proteins that bind free hemoglobin (Hb) to form complexes that are metabolized in
the reticuloendothelial system. Hb-haptoglobin complexes preserve the body´s stores of iron for reutilization.
- increased: acute and chronic inflammation
- decreased: liver disease, hemolytic anemia

Beta-1 globulins
Transferrin is a major iron-transporting protein in plasma. It transports iron from the intestine and between sites of
synthesis and degradation of hemoglobin.
- increased: iron-deficiency anemias
- decreased: liver diseases, malnutrition, inflammatory conditions, malignance

Beta-1/beta-2 interzone
Beta-lipoproteins (= low density lipoproteins, LDL) participate in transport of cholesterol.
- increased: hyperlipidemias
- decreased: starvation

Beta-2 globulins
C3 fraction of complement is a component of the complex system of serum proteins involved in inflammatory
reactions.
- increased: acute stress of organism
- decreased: active stages of autoimmune diseases (formation of immunocomplexes)

Gamma globulins are immunoglobulins synthesized in plasma cells; their are mainly of IgG class. Ig M is found
around the „start“ of electrophoresis.
- increased: polyclonal (or monoclonal) gammaglobulinemia, liver disease, chronic infections
- decreased: small children, hypoimmune syndrom
Monoclonal immunoglobulins (paraproteins) can be found in a different position: between alpha-2 and gamma
fraction. These proteins are synthesized by beta-cells during multiple myeloma or leucemia.

Normal values for individual major electrophoretic serum protein zones:

fraction %
albumin 56 - 66
alpha-1 globulins 2-3
alpha-2 globulins 8 - 12
beta-1 globulins 7 - 10
beta-2 globulins 3-6
gamma globulins 10 - 18

Using of electrophoresis in clinical practice

Serum protein electrophoresis is a simple technique that is helpful in establishing a diagnosis of monoclonal
gammopathies, liver cirrhosis, renal failure, hypogammaglobulinemia, and so on.

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 An „immediate response“ that occurs with stress or inflammation caused by infection, injury or surgical trauma
shows decrease of albumin and increase of alpha-1and alpha-2 globulin fractions (acute phase proteins are
selectively increased).

A „late response“ is correlated with infection and shows an increase in the gamma globulin band due to an
increase in immunoglobulins; albumin is decreased and alpha-2 globulin band is increased.

An electrophoretic pattern with extremely low intensity of gamma fraction (hypogammaglobulinemia) is observed
when an immunosuppressive disease is present.

In hepatic cirrhosis there is a broad elevation of the gamma globulins with reduction of albumin (polyclonal
gammopathy).

Monoclonal gammopathies are due to the clonal synthesis of a unique immunoglobulin and give rise to a sharp
gamma globulin band.

Nephrotic syndrome shows a selective loss of lower molecular weight proteins: the pattern shows a decrease in
albumin (65 kDa) and gamma globulin bands, but a retention of the alpha-2 band composed of the higher
molecular protein (alpha-2 macroglobulin, 725 kDa).

Hemolytic serum shows fraction of hemoglobin: increased beta band.

Literature:

● Engliš, M.: Novinky v medicíně 55: Interpretace elektroforézy plazmatických bílkovin v agarózovém gelu,
Avicenum, Praha, 1992 ISBN 80-201-0170-5
● Tichý, M.: Laboratorní analýza monoklonálních imunoglobulinů (paraproteinů), 1997 ISBN 80-902022-1-7
● Manuál: Elektroforéza bílkovin - SEBIA K20

REQUIRED KNOWLEDGE

Principle of electrophoresis, using electrophoresis in medicine, plasma proteins - classification, function,

concentration, difference between plasma and serum