M.PRASAD NAIDUMsc Medical Biochemistry,

Ph.D Research scholar.

Introduction

SERUM PROTEINS Composition Albumin: Conc. 60%, M.W. 69000, 585 AAs with 17

disulphide bonds.1. Synthesized from liver.2. Maintains colloidal osmotic pressure.3. Decreasing causes edema.4. Serves as asource of AA5. Decresed Alb –cirrhosis,nephrotic syndrome,

malnutrition.6. Increased alb - dehydration

Globulins1. It’s a glycoprotein with m.w. 90000 – 130000.

2. Types.

3. The α & β helps to transport proteins, hormones, vitamins, minerals and lipids.

4. γ globulins functions as immunoglobulins.

Total proteins - 6-8 gm/dl (100%)

Alb - 3.5 – 5 gm/dl (60%)

Glob - 2.5 – 3 gm/dl (40%)

α1 - 3%

α2 - 11%

β - 11%

γ - 15%

A:G Ratio: 1.5:1.

METHODS FOR SEPARATION OF SERUM PROTEINS1. Precipitation by salts.2. Cohn’s fractional precipitation method.3. Sedimentation by ultracentrifugation.4. Paper chromatography.5. Electrophoresis.

ELECTROPHORESIS Definition: Electrophoresis is the migration of

charged molecules in an electric field. The negative charged particles (anions) moves towards positive charged electrodes (anode). Positively charged particles (cations) moves towards cathod (negatively charged electrode).

Types: 1. Depending upon the nature of supporting medium

a. Agar gel electrophoresis (AGE).b. PAGE, SDS PAGE, QPNC PAGE (Quantitative preparative native continuous PAGE).c. Cellulose acetate electrophoresis.d. Capillary electrophoresis.

2. Depending upon the mode of technique.a. Slide gel electrophoresis.b. Tube gel electrophoresis.c. Disc electrophoresis.d. Low and high voltage electrophoresis.e. Two dimensional gel electrophoresis

Applications of Electrophoresis:1. Separating serum proteins for diagnostic purpose.2. Haemoglobin separation.3. Lipoprotein separation and identification.4. Isoenzyme separation and their analysis.5. Nucleic acid studies.6. Determination of molecular weight of the proteins.

FACTORS AFFECTING ELECTROPHORESISI. The electric field:a. Voltage - V ∞ Mb. Current - C ∞ Mc. Resistance- R 1/∞ M

II. The sample:a. Charge - C ∞ Mb. Size - S 1/∞ Mc. Shape - Molecules of similar size but different shape such as

fibrus

and globular proteins exhibit diffeent migration

characteristics. Because of the differential effect of

frictional and electrophoretic force.

III. The buffers:This determines and stabilizes the pH of the supporting medium and hence affects the migration rate of compound in a number of ways.

a. Composition: The buffer should be such that it does not binds with the compounds to be separated as this may alters the rate of migration. Therefore barbitone buffer is always preferred for the separation of proteins or lipoproteins.

b. Concentration: As the ionic strength of the buffThe er increases the proportion of current carried by the buffer will increase and the share of the current carried by the sample will decrease thus slowing down the rate of migration.

c. pH: For organic compounds pH determines the extent of ionization and therefore degree and direction of migration are pH dependent.

d. The supporting medium: The composition of supporting medium may cause adsorption, electro osmosis and molecular sieving. Which may influence the rate of migration of compounds. The commonly using supporting medium in the laboratory are agarose, polyacrylamide and cellulose acetate membrane.

Types of buffers used in electrophoresis1. Tris buffer.2. Glycine buffer.3. Sodium barbituric acid.4. TAE buffer (Tris acidic acid EDTA).

Types of StainsFor serum proteins – Amido block

- Coomassie brilliant blue

For isoenzymes - Nitro tetra zolium blueFor lipoprotein zones- Fat red 7B

- Oil red O- Sudan block B

For DNA fragments- Ethidium bromideFor CSA proteins - Silver nitrate

What is needed?Agarose - a

polysaccharide made from seaweed. Agarose is dissolved in buffer and heated, then cools to a gelatinous solid with a network of crosslinked molecules

Some gels are made with acrylamide if sharper bands are required

Buffer - in this case TBE

The buffer provides ions in solution to ensure electrical conductivity.

Not only is the agarose dissolved in buffer, but the gel slab is submerged (submarine gel) in buffer after hardening

Also needed are a power supply and a gel chamber

Gel chambers come in a variety of models, from commercial through home-made, and a variety of sizes

A gel being run

Agarose block

Positive electrode

DNA loaded inwells in the agarose

Black backgroundTo make loading wells easier

Comb

Buffer

The comb is removed, leaving little ‘wells’ and buffer is poured over the gel to cover it completely

The serum samples are mixed with a dense loading dye so they sink into their wells and can be seen

The serum samples are put in the wells with a micropipette.

Micropipettes have disposable tips and can accurately measure 1/1,000,000 of a litre

Pulsed field gel electrophoresis

Pulsed Field Gel Electrophoresis (commonly

abbreviated as PFGE) is a method for separating

large DNA molecules, which may be used for

genotyping or genetic fingerprinting.

Under normal electrophoresis, large nucleic

acid particles (above 30-50 kb) migrate at

similar rates, regardless of size. By changing the

direction of the electric field frequently, much

greater size resolution can be obtained

Pulsed field gel electrophoresis

SDS-PAGE

SDS-PAGE, officially sodium dodecyl sulfate

polyacrylamide gel electrophoresis, is a

technique used in biochemistry, genetics and

molecular biology to separate proteins

according to their electrophoretic mobility .

Quantitative preparative native continuous

polyacrylamide gel electrophoresis (QPNC-

PAGE) is a new method for separating native

metalloproteins in complex biological matrices.

Gel Electrophoresis

CAPILLARY ELECTROPHORESIS

MODES OF CAPILLARY ELECTROPHORESIS

TABLE 1

Indications for Serum Protein Electrophoresis

Suspected multiple myeloma, Waldenström's macroglobulinemia, primary amyloidosis, or related disorder

Unexplained peripheral neuropathy (not attributed to longstanding diabetes mellitus, toxin exposure, chemotherapy, etc.)

New-onset anemia associated with renal failure or insufficiency and bone pain

Back pain in which multiple myeloma is suspected

Hypercalcemia attributed to possible malignancy (e.g., associated weight loss, fatigue, bone pain, abnormal bleeding)

Rouleaux formations noted on peripheral blood smear

Renal insufficiency with associated serum protein elevation

Unexplained pathologic fracture or lytic lesion identified on radiograph

Bence Jones proteinuria

NORMAL PATTERN OF SERUM ELECTROPHORESIS1

i

γ-globulin

origin β2-globulin

β1-globulin

α2-globulin

α1-globulin

albumin

ABNORMAL PATTERN OF SERUM ELECTROPHORESIS

MULTIPLE MYELOMAMULTIPLE MYELOMA

i

Extra M-Band is seen Albumin

NEPHROTIC SYNDROME

i

Β-globulin and α2-gloublin Albumin

AGAMMAGLOBULINEMIA

i

Absence or decrease of γ-globulin and others normal

LIVER DISEASES

i

γ β -globulin

origin α2-globulin

α1-globulin

albumin

Characteristic Patterns of Acute-Reaction Proteins Found on Serum Protein Electrophoresis and Associated Conditions or Disorders

Increased albuminDehydrationDecreased albuminChronic cachectic or wasting diseasesChronic infectionsHemorrhage, burns, or protein-losing enteropathiesImpaired liver function resulting from decreased synthesis of albuminMalnutritionNephrotic syndromePregnancyIncreased alpha1 globulinsPregnancyDecreased alpha1 globulinsAlpha1-antitrypsin deficiencyIncreased alpha2 globulinsAdrenal insufficiencyAdrenocorticosteroid therapyAdvanced diabetes mellitusNephrotic syndromeDecreased alpha2 globulinsMalnutritionMegaloblastic anemiaProtein-losing enteropathiesSevere liver diseaseWilson's disease

Increased beta1 or beta2 globulinsBiliary cirrhosisCarcinoma (sometimes)Cushing's diseaseDiabetes mellitus (some cases)HypothyroidismIron deficiency anemiaMalignant hypertensionNephrosisPolyarteritis nodosaObstructive jaundiceThird-trimester pregnancyDecreased beta1 or beta2 globulinsProtein malnutritionIncreased gamma globulinsAmyloidosisChronic infections (granulomatous diseases)Chronic lymphocytic leukemiaCirrhosisHodgkin's diseaseMalignant lymphomaMultiple myelomaRheumatoid and collagen diseases (connective tissue disorders)Waldenström's macroglobulinemiaDecreased gamma globulinsAgammaglobulinemiaHypogammaglobulinemia

Differential Diagnosis of Polyclonal Gammopathy

InfectionsViral infections, especially hepatitis, human immunodeficiency virus infection, mononucleosis, and varicellaFocal or systemic bacterial infections, including endocarditis, osteomyelitis, and bacteremiaTuberculosisConnective tissue diseasesSystemic lupus erythematosusMixed connective tissueTemporal arteritisRheumatoid arthritisSarcoidLiver diseasesCirrhosisEthanol abuseAutoimmune hepatitisViral-induced hepatitisPrimary biliary cirrhosisPrimary sclerosing cholangitis

MalignanciesSolid tumorsOvarian tumorsLung cancerHepatocellular cancerRenal tumorsGastric tumorsHematologic cancers (see below)Hematologic and lymphoproliferative disordersLymphomaLeukemiaThalassemiaSickle cell anemiaOther inflammatory conditionsGastrointestinal conditions, including ulcerative colitis and Crohn's diseasePulmonary disorders, including bronchiectasis, cystic fibrosis, chronic bronchitis, and pneumonitisEndocrine diseases, including Graves' disease and Hashimoto's thyroiditis 

Characteristic Features of Monoclonal Gammopathies

Disease Distinctive features

Multiple myeloma M protein appears as a narrow spike in the gamma, beta, or alpha2 regions.M-protein level is usually greater than 3 g per dL.Skeletal lesions (e.g., lytic lesions, diffuse osteopenia, vertebral compression fractures)

are present in 80 percent of patients.Diagnosis requires 10 to 15 percent plasma cell involvement on bone marrow biopsy.Anemia, pancytopenia, hypercalcemia, and renal disease may be present.

Monoclonal gammopathy of undetermined significance

M-protein level is less than 3 g per dL.There is less than 10 percent plasma cell involvement on bone marrow biopsy.Affected patients have no M protein in their urine, no lytic bone lesions, no anemia, no

hypercalcemia, and no renal disease.

Smoldering multiple myeloma M-protein level is greater than 3 g per dL.There is greater than 10 percent plasma cell involvement on bone marrow biopsy.Affected patients have no lytic bone lesions, no anemia, no hypercalcemia, and no renal

disease.

Plasma cell leukemia Peripheral blood contains more than 20 percent plasma cells.M-protein levels are low.Affected patients have few bone lesions and few hematologic disturbances.This monoclonal gammopathy occurs in younger patients.

Solitary plasmacytoma Affected patients have only one tumor, with no other bone lesions and no urine or serum abnormalities.

Waldenström's macroglobulinemia IgM M protein is present.Affected patients have hyperviscosity and hypercellular bone marrow with extensive

infiltration by lymphoplasma cells.

Heavy chain disease The M protein has an incomplete heavy chain and no light chain.