SCHOOL OF HEALTH SCIENCES

2011/2012GTU 310 – CLINICAL BIOCHEMISTRY

PRACTICAL 1 – 6 MARCH 2012

Estimation of Albumin by Bromocresol green (BCG) Dye Method

Estimation of Total Protein by Biuret Method

Separation of Serum Proteins by Electrophoresis

NAME: MAISARAH AB SAMAD

MATRIC NUMBER: 105105

GROUP: 6

PROGRAM: BIOMEDICINE 3

LECTURER’S NAME: DR YUSMAZURA BT ZAKARIA

PRACTICAL 1A: ESTIMATION OF ALBUMIN BY BROMOCRESOL GREEN (BCG) DYE METHOD

OBJECTIVE

1. To estimate the albumin in serum by BCG Dye method

2. To record the optical density (absorbance) using the calorimeter and calculate the

concentration of the test sample from the formula.

INTRODUCTION

Serum proteins are proteins found in blood plasma. Albumin consists of

approximately 60% of the total proteins in the body while the other major part consists of

globulin. Albumin is synthesized in the liver and it is made up only of amino acids, without

prosthetic groups or other additives. Albumin is essential for maintaining the osmotic

pressure by regulating the blood oncotic pressure. A balance osmotic pressure is needed for

proper distribution of body fluids between intravascular compartments and body tissues.

Besides that, it also acts as a plasma carrier by non-specifically binding

several hydrophobic steroid hormones and as a transport protein for hemin and fatty acids. It

acts as carrier for many cations and water insoluble substances, like calcium and bilirubin.

Hypoalbuminemia is often associated with liver diseases, malnutrition or malabsorption,

burns or dermatitis, kidney and intestinal diseases. Hyperalbuminemia is rarely seen and is

usually associated with dehydration.

Bromocresol green (BCG) dye method is a method to determine albumin in serum or

plasma. Albumin will bind with the dye Bromocresol Green in buffered medium at pH 4.2 to

form a green coloured complex. The intensity of the colour of the resulting albumin-BCG

complex is directly proportional to the amount of albumin present in the sample.

PROTOCOL

Reagents and Apparatus:

1. Standard (Human serum albumin); Concentration: 46.75 g/L

2. BCG reagent; contain bromocresol green dye, succinate buffer pH 4.2, Brij-35

3. Pipettes

4. Spectrophotometer

5. Cuvetts

6. Vortex

Procedure:

The protocol of the procedure is described below.

1. The test tubes were labeled as ‘B’ for Blank, ‘S1’ for standard and ‘T1’, ‘T2’ and

‘QC’ for Test samples.

2. 3.0 ml of BCG reagent was added into each tube.

3. A micropipette was used to add 10µl of standard and test sample to the corresponding

test tubes. 10µl of distilled water was added in ‘Blank’.

4. The tubes were mixed gently using a vortex and the tubes incubated at room

temperature for 5 minutes.

5. The samples were read at 630nm against the blanks using a spectrophotometer.

6. The concentration of the albumin present in the given test sample was calculated

using the formula

Concentration of Albumin in sample = Atest/QC

Astandard

RESULT

BlankStandar

dTest (T1) Test (T2) Q.C

Optical

density

1 0 0.520 0.487 0.422 0.501

2 0 0.519 0.484 0.423 0.503

Average 0 0.5195 0.486 0.4225 0.502

Concentration

of albumin (g/L)0 46.75 43.69 37.98 45.18

Concentration range of QC: 38.7 – 61.9 g/L

DISCUSSION

X Concstd

The normal range of serum albumin is 35g/L to 50g/L. Based on the result above, the

concentration of the standard is 46.75 g/L, for T1 and T2; the concentrations are 43.69 g/L

and 37.98 g/L respectively. The concentration of QC is 45.18 g/L. Internal QC acts as quality

control in determining whether the method is acceptable. The range of concentration of QC is

38.7 to 61.9 g/L. In this practical, the concentration of QC is within the range, thus the

concentration of T1 and T2 are acceptable. The concentration of serum albumin in both T1

and T2 are normal.

Bromocresol green (BCG) dye-binding method for the quantitative determination of

serum-albumin was introduced in 1964 by DELANEY (Proc. Australian Assoc. Clin.

Biochem 64 (1964),1). Serum is diluted with buffered BCG at pH 7.0 and the decrease in

absorbance at 615 nm is measured. This change in absorbance is linear with albumin

concentration up to 50 mg/ml and other electrophoretically separated proteins like

hemoglobin and bilirubin do not interfere. This method is quicker compared to Biuret method

in analysing the serum protein. However, the measurement if albumin can be limited as it can

be interfered by haemolysed and lipaemic sera. Thus, in collecting the specimen, it is

important to avoid excessive haemolysis since every 100mg/dl of haemoglobin corresponds

to about 100mg/dl of albumin.

Abnormality in serum albumin concentration is often associated with clinical

significances that lead to hypoalbuminemia and hyperalbuminemia. Hypoalbuminemia or

decrease in serum albumin may due to reduced synthesis that caused by malnutriniton,

intestinal malabsorption, liver disease, wasting diseases, nephritic syndrome and

haemodilution. Hypoalbuminemia is also occurred due to excessive loss caused by nephritic

syndrome, massive burns, and protein-losing enteropathy. Severe burns that damage

capillaries and blood vessels cause a huge loss of serum proteins. The increased capillary

permeability caused by the burn damage allows a continual leakage of serum proteins out of

the vascular system. Analbuminaemia is a genetic disease with autosomal recessive in-

heritence that can lead to near to or absence of albumin. Besides that, serum globulin may

decrease due to haemodilution in shock, burns, haemorrhage. Serum globulin increases in

multiple myeloma, macroglobulinaemia, chronic liver disease, chronic infections and auto-

immune diseases. Increased albumin levels or hyperalbuminemia are generally not observed.

A rise in serum albumin may occur in dehydration due to haemoconcentration.

CONCLUSION

The intensity of the colour of albumin-BCG complex (green) is directly proportional

to the concentration of albumin present in the sample. The concentrations of albumin in both

Test samples are within the normal range and there might be no clinical significances

associated with the normal concentration.

PRACTICAL 1B: ESTIMATION OF TOTAL PROTEIN BY BIURET METHOD\

OBJECTIVE

1. To estimate the amount of protein present in the Test samples by biuret method.

2. To record the optical density (absorbance) using the calorimeter and calculate the

concentration of the test sample from the formula.

INTRODUCTION

Serum contains a large variety of proteins. Proteins are constituents of muscles,

enzymes, hormones and several other key functional and structural entities in the body. The

total proteins consist mainly of albumin and the various globulins. Proteins are essential in

the maintenance of the normal distribution of water between blood and the tissues.

Biuret method is use to estimate the total protein in serum. Cupric ions form chelates

with the peptide bonds of proteins in alkaline medium. A purple complex is formed as the

result of coordination of peptide nitrogen atoms with Cu 2+. The intensity of the violet colour

that is formed is proportional to number of peptide bonds which, in turn, depends on the

concentration of protein. The intensity of the colour can be measured by using a calorimeter

at 540nm.

PROTOCOL

Reagents and Apparatus:

1. Standard solution, S1; Concentration: 46.75 g/L

2. Test (Serum) sample solution (T1 and QC) containing unknown concentration of

protein

3. Biuret reagent (contains sodium potassium tartrate, cupric sulfate, sodium hydroxide,

potassium iodide)

4. Micropipettes 20µl

5. Cuvetts

6. Spectrophotometer

7. Vortex

Procedure:

1. The test tubes were labeled as ‘B’ for Blank, ‘S1’ for standard and ‘T1’, ‘T2’ and

‘QC’ for Test samples.

2. 1.0 ml of Biuret reagent was added into each tube.

3. A micropipette was used to add 20µl of standard and test sample to the corresponding

test tubes.

4. The tubes were mixed well by vortexing and were leaved at room temperature for 30

minutes at 25oC at room temperature.

5. The calorimeter was zeroed with the blank solution and the optical density (OD) or

absorbance was read at 540nm.

6. The results were tabulated and formula was used to calculate the concentration of

total protein.

Concentration of total protein = ODtest

ODstd

RESULT

BlankStandar

dTest (T1) Test (T2) Q.C

Optical

density

1 0 0.312 0.386 0.427 0.370

2 0 0.318 0.392 0.432 0.376

Average 0 0.315 0.389 0.4295 0.373

Concentration

of albumin (g/L)0 59.8 73.85 81.57 70.81

Concentration range of QC: 59.8 – 74.6 g/L

Normal Range

X ConcStd

Total Protein : 64g/L – 83g/L

Albumin : 35g/L – 50g/L

Globulins : 29g/L – 33g/L

A/G ratio : 1.2 to 1.5

DISCUSSION

The normal range of total protein is 64g/L to 83g/L. Based on the result above, the

concentration of the standard is 59.8 g/L, for T1 and T2; the concentrations are 73.85 g/L and

70.81 g/L respectively. The concentration of QC is 70.81 g/L. Internal QC acts as quality

control in determining whether the method is acceptable. The range of concentration of QC is

59.8 to 74.6 g/L. In this practical, the concentration of QC is within the range, thus the

concentration of T1 and T2 are acceptable. The concentration of total protein in both T1 and

T2 are normal.

Biuret method is one of protein quantification method in handling protein samples for

isolation and characterization and is a prerequisite step before submitting proteins for

chromatographic, electrophoretic or immunochemical analysis and separation. The name of

Biuret method is derived from the reaction between biuret and cupric ions in an

alkaline medium. A protein assay method should be chosen based on the sensitivity and

accuracy of method as well as the condition of the sample to be analyzed. Biuret total protein

reagent is the best choice if the total protein concentration in the samples is high (5 to 160

mg/ml). It is the most widely used calorimetric method; however it is not specific to the

components of the serum protein. It is the earliest colorimetric protein assay methods

developed for the determination of protein (Gornall, Bardawill, & David, 1949). Compounds

containing two or more peptide bonds react with the biuret reagent forming a purple colored

complex (Boyer, 2000). The colored product is the result of the coordination complex of

copper atom and two nitrogen atoms from each peptide chain (Switzer & Garrity, 1999). 30

minutes are required for the colour to reach the maxiumem intensity, thus the method is a bit

time consuming. This method is also limited in which haemolysed and lipaemic sera will

interfere strongly with the measurement of proteins.

There are certain factors that cause physiologic changes of total prorein.

Haemoconcentration causes an increase in serum total proteins occurs in dehydration. An

increase may also occure in multiple myeloma, macroglobulinaemia, chronic infections,

chronic liver disease and auto-immune diseases. A decrease in serum total proteins may

results from heavy losses of proteins in urine as in nephritic syndrome. Protein

undernutrition, intestinal malabsorption and protein losing enteropathy may also lower the

serum total proteins. A decrease may also occur in shock, burns, crush injuries, and

hemorrhage. Increased levels of total protein is associated with cholelithiases, multiple

myeloma and liver cirrhosis.Breakdown of proteins, as in hyperthyroidism, un-treated

diabetes mellitus, wasting diseases may also lower the level of proteins in serum.

The concentration of globulins can also be calculated by subtracting the concentration

of total protein with concentration of albumin. The calculated ratio of albumin to globulins,

termed the A/G ratio. Normally, there is a little more albumin than globulins, giving a normal

A/G ratio is between 1.2 and 1.5. For Standard, the ratio is 3.58, for test 1 and test 2, the ratio

are 1.45 and 0.87 respectively. For QC, the ratio is 1.76.

A low A/G ratio may reflect overproduction of globulins, such as seen in multiple

myeloma or autoimmune diseases, or low albumin synthesis due to cirrhosis, or selective loss

of albumin from the circulation, as occurs with kidney disease. A high A/G ratio suggests

underproduction of immunoglobulin as may be seen in some genetic deficiencies and in

some leukemias. More specific tests, such as albumin, liver enzyme tests, and serum protein

electrophoresis must be performed to make an accurate diagnosis. Low A/G ratio for test 2

and high A/G ratio for standard and QC require further diagnosis. For test 1, the ratio is

within the normal range.

 

CONCLUSION

The intensity of the colour of protein + Cu2+ complex (blue-violet) is directly

proportional to the concentration of protein present in the sample. The concentrations of total

protein in both Test samples are within the normal range and there might be no clinical

significances associated with the normal concentration. However, the A/G ratio is abnormal

for the standard, test 2 and QC which requires further diagnosis as the condition might be

associated with clinical significances due to abnormal concentration of globulin.

QUESTIONS

1. Name the other methods by which total protein can be measured.

a. Z-scan technique (Dhinaa, A. and Palanisamy, P., 2010)

b. Bradford Protein assays (Bradford, M.M., 1976)

2. What is the importance of Reagent blank in a calorimetric assay?

Reagent blank is important in calibration of calorimeter. This solution contains only

the diluent used for making the standard solution. Reagent blank is important in

spectroscopy to negate the effects of Background. This is to assure that the readings

are exclusively due to the components that been measured, not due to extraneous

chemical in solution.

3. From the albumin and total protein value for each sample, calculated thea. Serum globulins: Total proteins – Albumin

Standard: Serum globulins: 59.8 - 46.75 = 13.05 g/L

Test 1:Serum globulins: 73.85 - 43.69 = 30.16 g/L

Test 2: Serum globulins: 81.57 – 37.98 = 43.59 g/L

QC:Serum globulins: 70.81 - 45.18 = 25.63 g/L

b. A/G Ratio: Albumin/GlobulinStandard:A/G Ratio: 46.75/13.05 = 3.58

Test 1:A/G Ratio: 43.69/30.16 = 1.45

Test 2:A/G Ratio: 37.98/43.59 = 0.87

QC:A/G Ratio: 45.18/25.63 = 1.76

PRACTICAL 1C: SEPARATION OF SERUM PROTEINS BY ELECTROPHORESIS

INTRODUCTION

Protein molecules in solution will possess a combination of negative and ositive

charges. The net charge on a molecule being dependent upon the balance of the types of

amino acids in the protein and the pH of the solution. In electrophoresis, the pH of the

solution is controlled by adjusting it with buffers. At the pH used in most methods, most

serum protein possess a net negative charge (anions), some more negative than others. When

an electrical current is conducted through the protein solution, the protein move toward

anode. Because the protein differ in the magnitude of their negative charge, and to same

extent because of their size differences, they move toward the anode at different rates. The

more bulky the protein, the less negatively charged molecules and move more slowly than

albumins, which are relatively small and more negative. The relative influence of size vs

charge depends upon the medium used for the electrophoresis: cellulose acetate is primarily

charge; with agarose gel, size assumes a greater role; with polyacrylamide gel size is most

important reseparating the fractions.

Regardless of medium, the net result of these charge and size differences in molecules

is the ability to separate the mixture of proteins into its fractions and also to identify the

components based upon the distances that they have moved during the time of the test.

Further, the various protein fractions can also be quantified by staining them and measuring

the intensity of the colour.

The principle behind the electrophoresis is the protein in solution at a specified pH

will separate from each other when exposed to an electrical current for a period of time.

PROTOCOL

Components of Electrophoresis System

1. Support media which is 0.5% agarose gel

2. Buffer system – Barbital buffer pH 8.3

3. Power pack, Electrophoresis apparatus

4. Serum protein fixative solution (Methanol), staining solution (AmidoBlack) and

destaining solution (10% acetic acid)

5. Other accessories requied are Sample Gel holder, Template (to load sample), filter

paper wicks, drying oven and incubator, pipettes, tips and Detection system

(densitometer) for quotation of protein fractions.

Procedure

Electrophoresis was run in batches on Deluxe electrophoresis chamber. Sera were

applied with the help of wire loop applicator. Agarose gel was used as a support medium.

Barbital buffer was set at pH 8.3. Buffer pH was checked with the help of a pH meter before

use in electrophoresis. Line of application was directed towards cathode, and current was

applied at 100 Volts for 45 minutes. Different protein fractions moved depending upon

charge, molecular weight and size. Fixation was performed by methyl alcohol 85% and

glacial acetic acid 10% for 5 min. Staining was done with the help of AmidoBlack in 3%

acetic acid for10 min, whereas clearing was done with 10% acetic acid for 20 min. Strips

were dried in hot air oven and densitometric scanning was done for quantification of different

protein fractions. The procedure is modified based on paper by Waqar Azim, Saad Azim,

Khalid Ahmed et al. (2004).

RESULT

Figure 1: Electrophoresis pattern

DISCUSSION

Serum protein electrophoresis is a simple, reliable and specific method of separating

different protein fractions. Based on the result, the lane 1 shows the electrophoresis pattern of

Albumin

γ-globulin

α2-globulin

β-globulin

α1-globulin

normal serum while both lane 3 and lane 4 shows the electrophoresis pattern of abnormal

serum. There are five classes of plasma proteins which are albumin, α1-globulin, α2-globulin,

β-globulin and γ-globulin. Albumin moves fastest due its low molecular weight, followed by

α1-globulin, α2-globulin, β-globulin and γ-globulin. γ-globulin is the slowest due to its high

molecular weight. By comparing the intensity of the each types of serum protein, albumin has

the highest intensity which indicates high concentration in serum, followed by γ-globulin.

The normal range for total serum protein is 6-8 g/dl. More than half of total serum protein

(3.5-5 g/dl) is accounted for by albumin synthesized in liver. The remaining total serum

globulin fraction (2-3.5 g/dl) is comprised mainly by alpha and beta globulins synthesized by

the liver, and by immunoglobulins in the gamma band synthesized by plasma cells.

Figure 2: Normal serum protein electrophoresis (home.comcast.net)

In lane 3, the abnormal pattern shows low intensity of γ-globulin which can be

assumed to low concentration of γ-globulin than normal. Lane 3 pattern can be associated

with clinical conditions such as hypercholesterolemia, iron deficiency anemia and multiple

myeloma. For lane 4 pattern, the γ-globulin has high intensity showing high concentration of

γ-globulin than normal. Lane 4 pattern can be associated with clinical conditions such as

rheumatoid arthrisis, systemic lupus erythomatosus, cirrhosis, chronic liver disease and

multiple myeloma.

Electrophoresis has proved to be very useful in subfractioning or separating different

types of proteins. it has turned out to be a very useful diagnostic tool. Electrophoresis proved

very useful when routine investigations were hard to determined. Correct monitoring of

buffer pH, voltage, equal quantity of test and control applied and skill of technologist is the

prime importance in improving the quality of strips and proper interpretation of results.

Figure 3: Electrophoretic pattern of different diseases

(Waqar Azim, Saad Azim, Khalid Ahmed et al., 2004)

CONCLUSION

Albumin is the plasma protein that moves fastest due to low molecular weight and has

high intensity due to high concentration of albumin in the blood serum. The abnormal

electrophoretic pattern is associated with different diseases based on physiologic or

pathologic changes of serum protein. Electrophrosis has become the main tool in separating

the components of protein serum.

QUESTIONS

1. Name the 5 classes of plasma proteins and write which one moves fastest and why.There are five classes of plasma proteins which are albumin, α1-globulin, α2-globulin, β-globulin and γ-globulin. Albumin moves fastest due its low molecular weight.

2. What happens when there is a very low level of albumin in blood?

Hypoalbuminemia causes a decrease in oncotic pressure which can lead to water

retention and subsequent swelling in the body. It also can lead to symptoms that affect

the muscles of the body such as muscle weakness, muscles fatigue or muscle

cramping. In addition, individual who has been diagnosed with hypoalbuminemia

might experience low appetite. Ascites; an excess fluid that collects in the space

between the abdominal wall and the abdominal organs might also be present. Pleural

effusion is also common in patient with hypoalbuminemia. It is defined as the

accumulation of fluid in the space that surrounds the lungs.

3. Name two clinical conditions that lead to Hyperalbuminemia and Hypoalbuminemia.

Hyperalbuminemia is generally not observed.

Hypoalbunimeia is associated with malnutrition and malabsorption syndrom,

4. What are the other ways by which serum proteins can be separated?

The other ways are high-voltage electrophoresis immunodiffusion,

immunoelectrophoresis, and chromatography (ion-exchange, reverse phase

chromatography)

5. Name two stains that binds to proteins.

Coomassie Brilliant Blue and silver.

6. What is the pH of buffer? What is the reason for choosing this pH?

The buffer pH is 8.3. The isoelectric point of most proteins is between pH 4.0 and 7.5.

In pH 8 to 9, proteins will take on a negative charge and migrate to the anode. Most

protein electrophoresis is performed at pH 8.3 so that the proteins become anions and

migrate to the positively charged electrode (anode).

7. Name the clinical conditions that lead to the abnormal serum pattern of lane 3 and 4.

Lane 3: hypercholesterolemia, iron deficiency anemia and multiple myeloma

Lane 4: rheumatoid arthrisis, systemic lupus erythomatosus, cirrhosis, chronic liver

disease and multiple myeloma

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