sanjeeva yarkala et al. journal of scientific …...madhukar rao. k et al., j. sci. res. phar. 2012,...

Madhukar Rao. K et al., J. Sci. Res. Phar. 2012, 1(3), 98-101

Journal of Scientific Research in Pharmacy 2012, 1(3) 98-101

Journal of Scientific Research in Pharmacy Research Article Available online through ISSN: 2277-9469

www.jsrponline.com

Purification and Immunological characterization of riboflavin binding protein from Eagle (Aquila hastate) and Domestic fowl (Gallus gallus); A comparative study

Madhukar Rao. K* and M.S.K, Prasad Department of Biochemistry, Kakatiya University, Warangal, A.P, India.

Received on: 26-08-2012; Revised on: 27-08-2012; Accepted on: 11-09-2012

ABSTRACT

Riboflavin binding protein (RfBP) was isolated from the eggs of Aquila hastate and Gallus gallus. The protein was purified in two steps, DEAE-

Sepharose ion exchange chromatography and gelfilteration on Sephadex G-100. The holoprotein had an absorption spectrum characteristic of flavoproteins.

The purity of the protein was judged by SDS-PAGE technique. A single band on the slab and cylindrical gels revealed that the protein was pure. Comparison

of the mobility of RfBP with that of the s tandard molecular weight marker proteins suggested that RfBP from the egg white and yolk of Aquila hastate had a

molecular weight close to 29 Kd. Ouchterlony double diffusion analysis revealed that the antibodies raised against eagle egg RfBPs failed to cross react

with RfBPs isolated from hen eggs, suggesting immunological variation between t he RfBPs from these two birds.

Keywords: Riboflavin binding protein (RfBP), DEAE-Sepharose, Electrphoretic characterization, Molecular weight. Immunodiffusion analysis.

INTRODUCTION

Riboflavin is unique among the water soluble vitamins in that egg, milk and dairy products make the greatest contribution to its intake in Indian diets. Meat and fish are also good sources of ri boflavin and certain fruits and vegetables especially dark-green vegetables contain reasonably high concentrations. Animals are incapable of synthesizing the isoalloxazine skeleton of riboflavin .Hence this vitamin is required in the range of 2 to 10µg/g in the diet [1]. Biochemical signs of depletion arise within only a few days of dietary deprivation. Poor riboflavin status in western countries also seems to be of most concern for the elderly and adolescents. Riboflavin deficiency has been implicated as a ri sk factor for cancer.

Recent studies have established that vitamin binding proteins form a special group of soluble proteins present in eggs and other body fluids which ensure optimal bioavailability of the vitamins during growth and development. The indispensability of RfBP was demonstrated in a study on the homozygous recessive mutant (rd-rd) of domestic foul wherein, a gene mutation leading to the absence of RfBP resulted in the death of the developing embryos . Further the discovery that immunoneutralization of RfBP in animals such as rats and monkeys resulted in the abrupt termination of pregnancy clearly established the functional significance of RfBP [2].

The present investigation was carried out to isolate and characterize RfBP from the eggs of two different birds. Hen (Gallus gallus) and Eagle (Aquila hastate). For a comparative study Aquila hastate belongs to rail family Accipitridae, an eagle may resemble a vulture in build and flight characteristics but has a fully feathered (often crested) head and strong feet equipped with great curved talons. The Indian eagle is about 60 cm in length and has a wingspan of 150cm. It is broad-headed, with the widest mouth of all eagles.

RfBP was first isolated from the hen egg white [3-6] and recently from Emu (Dromaius novaehollandiae) egg-white [7]. These methods were slightly modified for the isolation of egg white and yolk

RfBPs from Aquila has tate.

MATERIALS AND METHODS

Aquila hastate eggs were procured from Old-city, Hyderabad, Andhra Pradesh, and Hen eggs were procured from local Hunter road Hanamkonda. The white and yolk were separated and used immediately

or stored at -120C. DEAE- Sepharose and Sephadex G-100 were obtained

*Corresponding author: Madhukar Rao Kudle Department of Biochemistry, Kakatiya University, Warangal, A.P, India. Mobile: 09985568554. *E-Mail: [email protected]

from Sigma Aldrich Chemical Company. St. Louis, USA. Bovine Serum albumin, acrylamide, N, N, N1, N1- Tetramethylethylene- diamine, N, N1-methylene-bis-acrylamide and SDS were procured from Loba Chemical,

Bombay, India.

Isolation and purification of eagle egg and hen egg white & yolk riboflavin binding protein (RfBP):

RfBP from eagle egg white and yolk was isolated following the methods previously reported [8 , 9] with a few modifications. Eagle egg white or yolk was collected and homogenized with an equal volume of 0.1 M sodium acetate buffer pH 4.5. To the clear supernatant DEAE- Sepharose previously equilibrated with 0.1 M sodium acetate buffer pH 4.5 was added. The DEAE- Sepharose with bound protein was washed with an excess of 0.1 M sodium acetate buffer pH 4.5. Bound proteins were eluted with the same buffer containing 0.5 M sodium chloride by suction filtration. Fresh DEAE- Sepharose previously equilibrated with 0.1M sodium acetate buffer pH 4.5 was packed into the column and then the partially purified RfBP was loaded onto the column. Riboflavin binding protein was eluted from the column with 0.1 M sodium acetate buffer, pH 4.5 containing 0.5 M sodium chloride. Fractions were collected and absorbances were measured at 280 nm and 455nm. Further purification of eagle egg white and yolk RfBPs were achieved by gel filtration column chromatography using Sephadex G-100. The almost pure eagle egg yolk RfBP was loaded onto the column previously equilibrated with 0.02 M phosphate buffer pH 7.3 containing 0.5 M sodium chloride and eluted with the same buffer. Fractions were collected and the protein in each fraction was determined by the method of [10]. The same steps were followed for purification of RfBP from Hen

(Gallus gallus)&Coot (Fulica atra) egg white and yolk.

Polyacrylamide Gel Electrophoresis: SDS-PAGE was carried out according to the method of [11]

using sodium phosphate buffer containing SDS. The following were made. 1. Sodium phosphate stock buffer (pH 8.0): To 461ml of 0.2M sodium

hydroxide, 500 ml of 0.2M sodium dihydrogen phosphate was added and made up to 1 litre with distilled water. To this 5 mM EDTA, and 1g SDS were added.

2. Electrode Buffer: 500 ml of stock buffer was diluted to 1 litre with distilled water.

3. Acrylamide - Bisacrylamide Solution: 30g of acrylamide and 0.8 g of bisacrylamide were dissolved in 100ml degassed water.

4. Ammonium persulphate solution: 150 mg of ammonium persulphate was dissolved in 100 ml degassed water.

5. Sample Buffer: 20 ml of the electrode buffer was degassed and 600 mg of SDS was added to 1 ml of this buffer 30 mg SDS, 500 mg sucrose, 20µl β- mercaptoethanol and 20µl bromophenol blue were added.



6. Protein staining solution: Coomassie blue (0.2 g) was dissolved in a solution containing 50 ml methanol, 7 ml of acetic acid and 43ml water.

7. Destaining solution: The gels were destained with the solutions

containing 50% methods and 7% Acetic acid.

SDS-PAGE: Cylindrical Gel: The gels were prepared by mixing 2 ml distilled water 8 ml

running buffer, 4 ml acrylamide bisacrylamide solution, 20µl TEMED AND 2 ml ammonium persulphate solution. The samples were dissolved in 50µl of sample buffer. The samples were heated for 2 min. in a boiling water bath. 20 µl of the sample was loaded onto the gel tubes. The electrophoresis was carried out at 2-5 mA/tube until the dye reached the end of the tube. The gels were stained overnight with stainiong solutions, and after destaining the gels were stored in distileed water till they were photographed.

SDS-PAGE: Slab Gel: The gel were prepared by mixing 4 ml of distilled water,16 ml

electrode buffer, 8 ml acrylamide-bisacrylamide, 40 µl TEMED and 4 ml ammonium per sulphate. The prepared gel solution was poured into glass plates (14x14 cm) separated by 1 mm thick spacer. The samples were dissolved in 50 µl sample buffer and kept in a boiling water bath for 2min. The A. hastate and hen egg white and yolk samples (20µl) were loaded into the slots. The reaming gap was filled with the electrode buffer. The glass plates were fixed to the electrophoresis apparatus without disturbing the samples. The upper and the lower electrode chamber were filled with the electrode buffer. The electrode chambers were connected to the power supply. Initially electrophoresis was carried out at 15mA for 30 min, after which the current was raised to 30mA. Current supply was terminated when the tracking dye reached the end of the gel. The plates were removed from the chambers and the gel was removed from the glass moulds by flushing buffer between the plates. The gel was stained immediately at room temperature. Later the gels were destained using the destaining solution. Proteins were also stained by the silver staining method using a silver staining kit (Bangulore Genei Ltd).

Production of antibodies against riboflavin binding protein: Antibodies against eagle and hen egg white RfBPs were

produced adopting the method of [12]. Briefly the protein was emulsified with an equal volume of Freunds comlete adjuvant (Sigma) and injected subcutaneously at weekly intervals for 4 weeks into ra bbits at multiple sites .The rabbit were then bled through the ear veins 7 days after the completion of the booster dose. The presence of antibodies in the serum was tested using ouchterlony double diffusion analysis.

Ouchterlony double diffusion analysis was carried out as follows; Agarose plates (1.2%) were prepared in 0.05M sodium phosphate buffer pH7.8 containing 0.9% NaCl. The antiserum was placed in the central well and the proteins dissolved in the same buffer were placed in the adjacent wells. The appearance of precipitin lines indicated

the presence antibodies.

RESULTS AND DISCUSSION

The purified RfBP containing peak fraction obtained from Sephadex G-100 gel filtration column chromatography was used to record the absorption spectrum using UV-Visi ble recording spectrophotome ter. The spectral characteristi cs of Riboflavin are altered upon binding to the apo RfBP. These spectral changes are characterized by a red shift of the 450nm band which accompany the appearance of shoulders and a remarkable hypochromism at the 370nm band (Figs.1, 2a & 2b). The holoprotein showed absorption maxina at 375nm and 458nm in agreement with the data reported by [13].

Electrophoresis on analytical polyacralamide gels (7.5%) was conducted at pH 8.3. The purity of the isolated protein was judged by slab and cylindrical SDS-PAGE methods. The electrophoretic pattern obtained was shown in Fig. 3. A major band corresponding to RfBP along with few minor bands were seen in the DEAE-Sepharose eluted fraction. Complete purification was achieved by gel-filtration chromatography on Sephadex G-100, as a single band free from other minor contaminating proteins was obtained (Fig. 3&4). RfBP moved as a single band on the cylindrical gels also (Fig. 5). Comparison of the mobility of RfBPs with that of the standard molecular weight marker proteins revealed that the RfBPs had a molecular weight close to 29 kilodaltons.

Analysis of carbohydrate compositi on of hen egg white RfBP revealed that the protein contains 10% hexosamine and 4% neutral sugars with a single sialic acid at the terminus of a highly branched oligosaccharide chain [16]. Hen egg yolk RfBP also contains both hexosamine and hexose as well as multiple sialice acid residues [14]. In the present study, the isolated RfBPs from Hen, Coot and Eagle eggs were analyzed using SDS PAGE and then subjected to glycoprotein specific silver staining. All these glycoproteins (Fig. 6), as revealed by the gel banding profiles, isolated from three birds eggs had the the same electrophoretic mobility with an approximate molecular mass of 29 kDa.

Studies on the immunological aspects of RfBPs are very limited and a detailed analysis is needed. RfBP is immunologically a multideterminant antigen consisting of at least six immmunodominant regions each eliciting distinct site specific antibodies [15]. In the present study, the Ouchterlony immunodiffusion analysis revealed that the antibodies raised against eagle egg white and yolk RfBPs could cross react with eagle egg white and yolk RfBPs only and failed to cross react with hen egg white and yolk RfBPs (Figs 7& 8). Absence of precipitin line formation against the RfBPs from hen eggs could be due to, some extent, differences in the antigencity and antigenic domains. This could be due to less conserved secondary and tertiary structures of these proteins. The present study clearly showed that RfBP from Aquila hastate (flying bird) had an electrophoretic mobility similar to that of hen (non flying bird) egg-white and yolk RfBP having a molecular weight close to that of the hen egg-white and yolk RfBP suggesting that this protein mostly remained unaltered in these species. However ,the lack of immunological cross reactivity as judged by Ouchterlony double diffusion analysis indicate some differences during evolutionary conservation of these proteins . Thus the present study while clearly establishing the similarities in the physiocochemical properties between hen egg RfBPs and eagle egg RfBPs the proteins might not be totally immunologically related.

Fig. 1: Absorption spectrum of F Riboflavin Binding Protein



Fig. 2a: Absorption spectrum of Eagle egg -white Fig. 2b: Absorption spectrum of Eagle egg-yolk

Riboflavin Binding Protein Riboflavin Binding Protein

EW EW EW EY EY EY PMW Crude DEAE G10 Crude DEAE G100

97,000 66,000 43,000 29,000 18,400 HW HY CW CY EW EY PMW

1 2 3 4 5 6 7

97,400 60,400 43,000 29,100

EW EW HW HW

DEAE G100 DEAE G100

1 2 3 4

Fig. 3: SDS Polyacrylamide Gel Electrophoresis pattern of Eagle egg

white and egg yolk RfBPs.

1. Eagle egg white crude

2. Eagle egg white DEAE Sepharose eluted fraction 3. Eagle egg white Sephadex G-100 fraction 4. Eagle egg yolk crude 5. Eagle egg yolk DEAE Sepharose eluted fraction 6. Eagle egg yolk Sephadex G-100 fraction 7. Protein molecular weight marker (20,000 to 97,400 kD)

Fig. 6: SDS Polyacrylamide Gel Electrophoresis pattern of Hen,

Coot, Eagle egg white and egg yolk RfBP’s Silver staining

1. Hen egg white Sephadex G-100 fraction 2. Hen egg yolk Sephadex G-100 fraction 3. Coot egg white Sephadex G-100 fraction 4. Coot egg yolk Sephadex G-100 fraction 5. Eagle egg white Sephadex G-100 fraction 6. Eagle egg yolk Sephadex G-100 fraction 7. Protein molecular weight marker (18,400 to 97,000 kD)

EW EY HW PMW G-100 G-100 G-100

97,400 60,400 43,000

29,100

Fig. 4: SDS Polyacrylamide Gel Electrophoresis pattern of Eagle egg

white and Hen egg white RfBPs.

1. Eagle egg white DEAE Sepharose eluted fraction

2. Eagle egg white Sephadex G-100 fraction 3. Hen egg white DEAE Sepharose eluted fraction 4. Hen egg white Sephadex G-100 fraction 5. Protein molecular weight marker (20,000 to 97,400 kD)

Fig. 5: Cylindrical Gel Electrophoresis (SDS

Polyacrylamide) pattern of Eagle egg white

and Hen egg white RfBPs.

1. Eagle egg white G-100 eluted fraction 2. Eagle egg yolk G-100 eluted fraction 3. Hen egg white G-100 eluted fraction 4. Protein molecular weight marker

(20,000 to 97,400 kD)

97,400

60, 400

43,000

29.100

Eew

G



Fig. 7: Ouchterlony double diffusion analysis Fig. 8: Ouchterlony double diffusion analysis (The central well contains Eagle egg white antiserum) (The central well contains Eagle egg yolk antiserum)

1. Purified Hen egg yolk RfBP Sephadex G-100 fraction 1. Purified Eagle egg white RfBP Sephadex G-100 fraction 2. Purified Hen egg white RfBP Sephadex G-100 fraction 2. Purified Hen egg yolk RfBP Sephadex G-100 fraction 3. Purified Eagle egg white RfBP Sephadex G-100 fraction 3. Purified Hen egg white RfBP Sephadex G-100 fraction 4. Purified Eagle egg yolk RfBP Sephadex G-100 fraction 4. Purified Eagle egg yolk RfBP Sephadex G-100

CONCLUSION

The present investigation was clearly showed those are correlated to both electrophoretic mobility and molecular levels of eagle and hen, and also physiocochemical properties be tween hen egg RfBPs and eagle egg RfBPs the proteins might not be totally immunologically related. That RfBP from Aquila has tate (flying bird) had an electrophoretic mobility similar to that of hen (non flying bird) egg-white and yolk RfBP having a molecular weight close to that of the hen egg-white and yolk RfBP suggesting that this protein mostly remained unaltered in these species. However ,the lack of immunological cross reactivity as judged by Ouchterlony double diffusion analysis indicate

some differences during evolutionary conservation of these proteins.

REFERENCE: 1. Dadd R.H. In comparative insect physiology Biochemistry and

Pharmacology (eds krekut, G.A. and Gillbert ,b.I.) Pergamon Press, New York,1985; 4: pp. 313-330.

2. Adiga PR, Visweswariah S, Karande A, Kuzhandhaivelu AN . J. Biosci., 1988: 13; pp 87-104.

3. Rhodes MB, Benntt N, Feeney RE. The flavor protein – Apoprotein system of Egg White. J. Biol. Chem., 1959: 234; 2054-2060.

4. Hamazume Y, Mega T, Ikenaka T. Yolk-riboflavin binding proteins and amino acid sequence of egg white riboflavin binding protein J. Biochem., 1984: 95; 1633-1644.

5. Miller M.S, Buss E.G, White H.B.III. Biochemistry, 1984; 23: 569-76.

6. Madhukar Rao K, PrasadM.S.K. Purification and Characterizationof riboflavin binding protein from (RfBP) Hen (Gallus gallus) DEAE-Sepharose column chromatography, Inter. J. of Appli. Biology, 2011: 2; 27-29.

7. Bindu Mary Rajan, Prasad M.S.K. Isolation and Purification of Riboflavin binding protein (RfBP) from the egg white of emu (Dromaius novaehollandiae). Iner. J. of Plant and Animal Scie., 2012: 2; p 5-9

8. Madhukar Rao K, Prasad M.S. K. Isolation and Characterization of Riboflavin binding protein from the egg white of Eagle (Aquila hastate). Inter. J. of Pharama. S cie. Reas., 2012a: 3; 494-496.

9. Madhukar Rao K, Prasad M.S.K. Isolation and Purification of Riboflavin binding protein from Eagle egg yolk (Aquila hastate). Intre. J. Appli. Biology, 2012b: 3; 351-354.

10. Lowry O.H, Rosebrough N.J, Farrl A.L, Randalll R. J. Protein measurement with the Folin phnol reagent . J. Bio Chem., 1951: 193; 265-275.

11. Lemmli U.K. Cleavage of structural protein during the assembly the head of bacteriophase T4 Nature., 1979: 227; 680-685.

12. Prasad M.S.K, Adiga P.R. Modulation of Prolaction Re ceptors in the Male rat. Ind.J. E xp.Bio., 1979: 17; 1166.

13. Choi J.D, McCormic D. B. Archives of Bio-Chemistry and bio-Physics, 1980: 204; 41-51

14. Ostrowski W, Skarzynski B, Zak Z. Biochem. Biophys. Acta., 1962: 59; 515-19.

15. Adiga P.R, Subramaniam S, Rao J, Kumar M.Hum. Reprod. Update. 1997: 3; 325-34.

16. Philips J.W. Ph.D. Thesis. 1969: Pennsylvania State University.

Source of support: Nil, Conflict of interest: None Declared

HY G-100

HW G-100 EW an

EWG100 EY G-100

EW G-100

HW G-100

EW an EWG100

EY G-100

sanjeeva yarkala et al. journal of scientific …...madhukar rao. k et al., j. sci. res. phar. 2012,...

Documents