RESARCH PROJECT

A ResearchProposal
On
BIOCHEMICAL STUDY OF SERUM PROTEINS
OF BUFFALO (Bubalus bubalis) and
IT’S COMMERCIAL UTILIZATIOM

Submitted by:
Sujan Gautam
Universal Science College, Maitidevi plaza,
Kathmandu Nepal

Submitted To:
Department of Biochemistry, Universal
Science
College, Maitidevi plaza,
Kathmandu, NEPAL

Principal investigator: Sujan Gautam

co-investigator: Vijayendra agrawal

Introduction

Blood and serum are frequently used as samples for proteomic analysis. Serum is a rich source of biochemical products that act as biomarkers of disease or reflect physiological status of a patient. Proteomic technologies are being used to discover and identify disease-associated biomarkers. The application of current proteomic technologies in the search for potential diagnostic/prognostic indicators in the serum of patients is limited by highly abundant albumin and immunoglobulins that constitute more than 60% of the total serum protein. Removal of abundant serum proteins will help in the discovery and detection of less abundant proteins that may prove to be informative disease markers (Chen et al., 2005). The concentration of plasma proteins is a useful clinical diagnostic tool and is often indicative of metabolic disorder.
Albumin is the most abundant protein in serum and is synthesized by the liver to maintain homostatic levels when protein intake is sufficient. Decreased albumin concentration, which is typically correlated with decreased total protein concentration, indicates protein malnutrition. Depending on the composition of the diet, decreased albumin may indicate energy malnutrition whereby proteins are catabolized for gluconeogenesis.
Immunoglobulin G (IgG) is the second most abundant protein in plasma and is synthesized by B-lymphocytes in response to the sum of immunogenic stimulation. The levels of IgG are decreased by (protein) malnutrition and elevated by infectious disease.
Acute phase proteins (APP) are an ensemble of plasma proteins synthesized by the liver in response to tissue damage and inflammation associated with traumatic and/or infectious disease. Transferrin (Tf) and fibrinogen (Fb) are the most abundant acute proteins that can be accurately measured. There are additional proteins that become detectable when transferrin and fibrinogen levels are increased. Increased IgG and acute phase proteins indicate an infection with concurrent tissue damage. Increased IgG without increased fibrinogen indicates immunological response to infection without concomitant decrease in tissue function (Acute phase protein, 15 June 2007).
Asian buffalo, Bubalus bubalis, has been heavily domesticated and thus is now widespread. The suspected native domain of B. bubalis was from central India to southern Nepal in the west to Vietnam and Malasiya in the east. In the wild, water buffalo are found in tropical and subtropical forests as well as wet grasslands (Water buffalo, 17 th June 2007). The buffalo population in Nepal can be broadly classified into three groups based on their breed characteristics- Hill buffalo, Terai buffalo and Indian breeds (Rasali, 2000). Lime, Parkote and Gaddi are the three breeds of Hill buffalo reported in the literature (Rasali and Crow, 1999).
Forward motility-promoting protein isolated from buffalo blood serum has the potential for the treatment of human infertility (due to low sperm motility) a great social curse in all human races. The product isolated by the present invention has also the potential for improving farm animal breeding with special reference to buffalo, a milch animal of great economic importance in many countries including India (Majumder et al., 2003).
This study is directed towards the isolation and purification of albumin from serum for its commercial utilization. In addition, study for the identification of some biomarker protein will be done to differentiate normal and diseased buffalo. As far as my knowledge, such a study hasn’t been conducted so far in Nepal.

Objectives:

. General objective:
· Study of serum protein from Buffalo.

ii. Specific Objectives:
· To isolate serum from blood sample.
· To isolate albumin from blood serum

a. By (NH4)2SO4 precipitation
b. By TCA/ acetone (or ethanol/ methanol/ DMSO) method
· To perform size exclusion chromatography to purify the albumin fraction.
· To check the purity of protein and also compare the pattern of proteins isolated from diseased as well as non-diseased serum samples by SDS-PAGE and find some biomarkers for the identification of disease one.
Materials and methods:
Chemicals and Reagents: Acetic acid, TCA, Acetone, Methanol, DMSO, Acrylamide, Bis-acrylamide, Ammonium persulphate, Ammonium sulphate, bis-Acrylamide, b-mercaptoethanol, Bromophenol blue, CBB-G 250 (Coomassie brilliant blue), CBB-R 250, Ethanol, Sucrose, Glycerol, Glycine, Phosphoric acid, Potassium dihydrogen phosphate, di-Sodium hydrogen phosphate, Sodium dodecyl sulphate, Hydrochloric acid, Bovine Serum Albumin (BSA), TEMED (Tetramethylene ethylenediamine), Tris (tris-hydroxymethyl aminomethane), Potassium chloride, Sephadex G-100.

Equipments: Analytical balance, Cold centrifuge, Fraction collector, Magnetic stirrer, Micropippetes, pH meter, Protein electrophoretic set, Spectrophotometer, Vaccum filter, Chromatographic column

I. Isolation of blood serum

· Buffalo blood will be collected both by venepuncture and by sacrifice of buffalo into plain collection tubes.
· The blood samples will be allowed to clot at room temperature for 30 minutes and then centrifugation will be done at 5000 rpm for 15 min to collect the serum.
· The amount of protein present in serum will be determined by Bradford method and SDS-PAGE will be done according to Laemmli.
· For the purification of albumin from serum, two approaches will be adopted: (NH4)2SO4 precipitation and TCA precipitation.

II. Purification of serum albumin by (NH4)2SO4 precipitation(Little and Little, 1994)

· Proteins present in serum sample will be precipitated using different percent saturation of ammonium sulfate. Precipitation will be done slowly over a period of 30-45 minutes with gentle stirring with the help of a magnetic stirrer to get the desired % saturation.

Take the serum
↓
Dilute the serum with 0.1 M phosphate buffer, pH 7.0 to get 1 mg /ml protein concentrati.


*The number of grams of ammonium sulfate to be added to 1 liter of solution at 20°C to get a desired saturation= 533(S2-S1)/100-0.3S2, where S1 is the initial % saturation and S2 is the final % saturation.
The ammonium sulphate precipitation containing albumin as a major fraction will be subjected to size exclusion chromatography for further purification.

III. Purification of serum albumin by 10% TCA in acetone/ ethanol/ methanol and DMSO (Chen et al., 2005).
Serum albumin will be precipitated by 10% TCA in different solvents viz. acetone, ethanol, methanol and DMSO and the purity of albumin fractions obtained will be accessed by SDS-PAGE. The fraction of maximum purity will be used for further purification of albumin by size exclusion chromatography.


Take 20 ml sample of buffalo serum and add four volumes of ice-cold acetone (or ethanol/ methanol/ DMSO) containing 10% w/v TCA.


Mix by gentle vortexing



Incubate the mixture at -200C for 90 min and centrifuge at 15 000 rpm, 4 0 C, for 10 min


Store the supernatant (a)


Then, add 1 mL of ice-cold acetone (or ethanol/ methanol/ DMSO) to wash the precipitate

Incubate the sample on ice for 15 min and then
Centrifuge at 15000 rpm/10min

Store the supernatant (b)


Mix supernatant (a) and (b) and add 1 ml of ice-cold acetone (or ethanol/ methanol/ DMSO) to the supernatant mixture to completely precipitate the proteins in the supernatant. Dissolve the protein precipitate in minimum volume of 0.1 M Phosphate buffer, pH 7.0.

IV. Purification of serum albumin by size exclusion chromatography (Andrews, 1965)

1. Prepare slurry of Sephadex G100 in 0.15 M KCl to get a bed volume of 50 ml. Mix required amount of gel beads with KCl and let the beads swell for 24 h.
2. Slowly pour the swelled beads in the column with outlet closed. Let the beads settle and then pass the KCl solution through the column at a flow rate of 0.5 ml/ min. Percolate the column with the same KCl solution 1-2 times so that column is well packed.
3. Equilibrate the column with 0.05 M Tris.Cl, pH 7.5 containing 0.1 M KCl.
4. Apply the protein sample on top of the column.
5. Elute the protein at a flow rate of 0.5 ml/ minute and collect 3 ml sample per tube.
6. Quantitate each eluted fraction by taking absorbance at 280 nm and pool the fractions showing peak value.
7. Dialyse the pooled fraction against 0.1 M Phosphate buffer, pH 7.0 containing 50% glycerol for 12 h with 2 changes of buffer.
8. Quantitate the protein content and do SDS-PAGE to access the purity of albumin purified.

V. Protein Quantitation by Bradford method (Bradford, 1976)

Mix Protein solution (0.1ml) with 4.9 ml of Bradford reagent. Vortex and incubate for 2 minutes. Measure absorbance at 595nm. Use BSA as a standard and Bradford reagent as blank.
(Bradford reagent: Dissolve 0.1 gm of CBB-G 250 in 50 ml of 95% C2H5OH and add 100 ml of 85% w/v H3PO4. Dilute the solution to 1L. Filter before use.)
VI. SDS-PAGE (Laemmli, 1970)
SDS-PAGE will be performed to check the purity of isolated albumin sample. It will also be used to compare the protein profile of normal and diseased buffalo serum so as to find some biomarker for the identification of diseased buffalo.
Expected outcome:
Albumin will be isolated and purified from the serum of buffalo blood. In addition, may be, some biomarker for the differentiation of normal and diseased buffalo can be found among the myriad of proteins present in serum.
References:
Acute phase protein. (15 June 2007) http://en.wikipedia.org/wiki/Acute_phase_protein

Andrews P (1965) The Gel-Filtration Behaviour of Proteins Related to their Molecular Weights over a Wide Range. Biochem. J. 96: 595

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254

Chen YY, Lin SY, Yeh YY, Hsiao HH, Wu CY, Chen ST, Wang AH (2005) A modified protein precipitation procedure for efficient removal of albumin from serum. Electrophoresis 26: 2117-2127

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the Head of Bacteriophage T4. Nature 227: 680-685

Little SE, Little JT (1994) Rapid Protease Screening Using Ammonium Sulfate Precipitations and the AvantiTM High Performance Centrifuge. Application Information A-1796A. Fullerton, CA, Beckman Instruments, Inc.
http://www.beckman.com/literature/Bioresearch/1796A(A).pdf

Majumder GC, Mandal M, Banerjee S (2003) Process for the purification of a new motility-promoting protein from buffalo serum: a slaughter house waste. http://www.freepatentsonline.com/6613737.html

Rasali DP (2000) Recent Trends in Buffalo Production in Nepal. In Buffalo Newsletter http://www.buffaloreview.20m.com/Leadarticle.html, pp 6-10

Rasali DP, Crow GH (1999) Production of buffaloes (Bubalus bubalis) in the mountains and hills of Nepal: Constraints and opportunities. In http://www.buffaloreview.20m.com/commentary.htm.

Water buffalo. (17 th June 2007) http://animaldiversity.ummz.umich.edu/site/accounts/information/Bubalus_bubalis.html


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