Protein Purification - Iowa State Universityduahn/teaching/Neobiomaterials and... · A major avian...

Protein Purification ‐ Separation of Egg Components

Proteins of Egg White

Ovotransferrin 

•

A major avian egg white protein (12-15% of total egg white protein)

•

A monomeric glycoprotein consists of a 686-residue single polypeptide chain. Molecular mass: ~ 78 kDa

•

pI: 6.73

for apo-form (iron-free), 5.78

for holo-form•

Iron binding protein (reversible, two iron atoms/molecule, as Fe3+).

•

Metal-free (apo) form is easily destroyed by physical and chemical treatments, while holo-form (iron bound) is a salmon-pink colored

•

Iron-complex is stable to proteolytic hydrolysis and thermal denaturation.

Important Characteristics for  Ovotransferrin Separation

• The release of Fe3+ from ferric transferrin requires the presence of a simple anion such as pyrophosphate, sulfate, and chloride

• Ovotransferrin is the most heat liable protein in egg white– Forms aggregation by heating at 60 C– Iron-binding increases the chemical and heat

resistance of ovotransferrin• pH is important for iron-binding• Bicarbonate is essential for iron binding

– Anion is needed for binding and release of iron

Ovotransferrin Separation Methods

• Precipitation of ovalbumin by shaking

• Ammonium sulfate precipitation

• Ion exchange chromatography– CM‐Cellulose cation exchange

– DEAE‐cellulose anion exchange

– Q‐Sepharose Fast Flow column

– Duolite C464 and C476) chromatography for industrial‐scale production 

• Immobilized metal affinity chromatography (a copper‐loaded Sepharose 6B 

column) from undiluted, blended egg white 

• A bi‐functional dye‐ligand chromatography – DEAE Affi‐Gel Blue, as the first step to fractionate egg white proteins. 

– Fast flow liquid chromatography for purification

• A two‐step chromatographic procedure – gel permeation on a Superose‐6 Prep Grade column

– anion‐exchange chromatography on a Q‐Superose Fast Flow

Molecular formulas of cellulose-based ion exchangers

Ion –

exchange Chromatography

New Separation Goals

• Should fit for use in human foods– Selection of solvent or chemicals are very important

• Scale‐up capability• Simple and economical

– Minimize sample volumes

• Maintain antimicrobial capabilities– Iron‐free form has stronger antimicrobial 

capabilities

Binding of Iron to Ovotransferrin

• Requires 1 molecule of HCO3‐

per atom of  Fe3+ 

• pH is important 

• Presence of anion is required

Egg white after diluting with water

After adding FeCl3 to saturate  ovotransferrin in egg white

Precipitation of egg white proteins using  ethanol ‐

43% 

Effect of pH on iron binding capacity of  ovotransferrin 

b

b

a a a a

0.0

2.0

4.0

6.0

8.0

10.0

12.0

Ovo

tran

sfer

rin c

onc.

(mg/

ml)

4 5 6 7 8 9pH

Iron saturation of ovotransferrin in egg  white solution

aa

b

c

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Abs

orba

nce

at 4

68 n

m

0.25 0.5 1 2 3

20 mM FeCl3.6H2O (ml)/100ml egg white*

c

*2 x diluted egg white solution, pH 9.0

Effect of pH on the yield of ovotransferrin from 2x‐ diluted egg white solution 

First extraction   

Second extraction 

Vol. x Conc.

pH  

Conc. 

Vol. 

Conc. 

Vol. 

total

Yield (%)

7 

9.40ab 

61.3a 

2.48b 

14.0ab 

610.7ab 

87.18

8  

8.94b 

61.7a 

2.82b 

13.7b 

581.4b 

83.07

9 

9.69a 

62.3a  

6.62a 

14.3a 

671.5a 

95.88 

The solution obtained from the first extraction with 43% ethanol. 

Conc: mg/ml; vol: ml

*The yields were calculated based on theoretical value (7.0 mg/ml) of ovotransferrin in 2x‐

diluted egg white solution. 

The yields of each supernatant obtained by adding 43%  ethanol to egg white solution

Extraction    Concentration 

Volume Vol. x Conc. 

Yield*

(mg/ml) 

(ml) 

(mg)

(%) 

First      

11.99 

43.95  527.30 

75.33

Second   

7.94 

22.32  170.56 

24.37

First Extraction: 43% ethanol extraction and then 59% ethanol precipitation. 

Second extraction: 64% ethanol was used to precipitate ovotransferrin. 

*The yields were calculated based on theoretical value (700 mg) of ovotransferrin 

in 100 ml of 2x‐diluted egg white solution.

Effect of ethanol concentration on the extraction of  holo‐OTF from egg white

Lane 1: 2x‐diluted egg whiteLanes 2~5: supernatants of 20%, 33%, 43% and 50% of ethanol extractionlanes 6~9: precipitates of 20%, 33%, 43%, and 50% of ethanol extraction. 

1        2      3       4        5       6       7       8     

9

43%

ethanol

Ovotransferrin

Ovoalbumin

Precipitation of extracted holo-OTF using ethanol

Lane 1: diluted egg white, Lanes 2 ~ 5: precipitates by 53%, 56%, 59% and 62% ethanol

extractionLanes 6~9: supernatant by 53%, 56%, 59%, and 62% ethanol

extraction

1      2        3      4         5     6       7      8      9

Ovotransferrin

Ovoalbumin

59% ethanol ppt 59% ethanol supernatant

http://www.activinlf.com/activinlf/ActivatedLactoferrinTech/alt_whatis.htm

Why should lactoferrin be activated?

Activation of Ovotransferrin

• Removal of iron from holo‐ovotransferrin– Bicarbonate is essential– A simple anion such as pyrophosphate, sulfate, and 

chloride is required in vitro– The anion‐induced Fe3+ release is closely related to 

the opening of a domain in either lobe– pH: citrate facilitate the release of iron

• Removal of iron from ovotransferrin solution– Chelex– AG ®1‐X2 resin (chloride form) 

Antimicriobial Agents and Chemotheraphy 1982. 21(5): 840-841

¥ : Free ovotransferrin + 50 mM NaHCO3¥ O : Ovotransferrin immobilized on

Spharose 4B + 50 mM NaHCO3

¥ 5 mg/ml of ovotransferrin

¥ Higher bacteriostatic activity of matrix-bound ovotransferrin might be ascribed tohigher stability of matrix-bound protein

Antibacterial activity against

E. coli

Iron Removal from Holo‐Ovotransferrin

0.1 0.2 0.3 0.6 0.9

aab

b

c

first

a aa

b

Second

0.00.51.01.52.02.53.03.54.0R

esid

ual i

ron

(ppm

)

AG1-X2 resin (g)/ 100 ml solution *

b

c

- Residual irons were determined by Ferrozine test- OTF concentration of the solution was around 6.5 mg/ml

The influence of citrate on the residual iron in  ovotransferrin solution

Protein 

Ovotransferrin 

Residual iron 

Treatment  (mg/ml)

(mg/ml) 

(ppm) 

Without

dialysis  

8.59  8.00 

0.52

Dialysis              

8.57         

8.42 

0.53

Residual iron: The amount of iron remaining in the solution after iron removal. 

Protocol for Ovotransferrin Purification

Dilution of egg white with 1 volume distilled water 

Add NaHCO3, NaCl, FeCl3 and adjust pH to 9.0

Stir for 30 min, add ethanol (43%, final), and centrifuge at 3,220 x g for 20 min

Supernatant (1st)             Precipitate

Re‐extract with 43% ethanol 

Centrifugation at 3,220 x g, 20min

Supernatant (2nd)

Purification method‐continued

Add ethanol (59%, final) and centrifuge

Dissolve precipitant with distilled water  

pH adjustment to 4.7 with 50 mM citric acid

Add

0.6 g of AG ®1‐X2 resin to 100 ml holo‐ovotransferrin solution

Stir for 1 hr and filter through Whatman #1 paper

Apo‐ovotransferrin

Freeze dry                  

The yield of final ovotransferrin using the  developed method 

Sample         Conc. 

Vol. 

Vol. x Conc. 

Yield 

(mg/ml) 

(ml)  (mg) 

(%)

Egg white        

7.00        

100      

700     

100                         

Final solution     

6.36  108 

693

99.03

The theoretical concentration of ovotransferrin in 2x‐diluted egg white solution used was 7 

mg/ml.        

Final solution: Apo‐ovotransferrin solution produced after iron removal. 

Purity of ovotransferrin

Lane 1: marker3: 2x diluted egg white4: Supernatant after ethanol extraction5: Supernatant after precipitation of holo-OTF 6: Precipitant

OTF

1    2    3    4      5     6

ovalbumin

200 kD

97 kD66 kD

45 kD

Summary

• Dilution of egg white and iron saturation of apo‐ ovotransferrin to holo‐form using FeCl3  

• Extraction of holo‐ovotransferrin by precipitating egg white  proteins with 43% ethanol

• Precipitation of extracted ovotransferrin with 59% ethanol

• Irons removal from holo‐OTF using AG1‐X2 resin

• Freeze drying of ovotransferrin

Advantages of the Developed Protocol

• Can be scaled‐up easily • Simple process

• Compatible to use in human foods

• Materials can be recycled 

Significance of using Ovotransferrin as  an Antimicrobial Agent

• Prevent cross contamination

• Extend self-life of food products by inhibiting growth of foodborne pathogen during processing or storage

• Developing an economical, large-scale preparation method of ovotransferrin from egg white will increase the value and use of

egg

• High possibility of becoming a GRAS material like lactoferrin

• A natural antibacterial agent

• No addition of any hazard material during separation

Lysozyme

•

Lysozyme constitutes approximately 3.5% of hen egg white 

•

Egg white lysozyme consists of 129 amino acid residues with a  MW of 14.4 kDa 

•

Binds to ovomucin, transferrin or ovalbumin in egg white

•

The thermal stability of lysozyme is partly due to its four  disulfide bonds

•

Highly stable in acidic solution

and heating at 100 C for 1‐2  minutes

•

In nature, found mainly as a monomer

but also exist as a  reversible dimer

•

The dimeric form

of lysozyme exhibits therapeutic, antiviral  and anti‐inflammatory properties

Lysozyme

•

Demonstrates antimicrobial activity against a  limited spectrum of bacteria and fungi 

•

Its enzyme activity can be enhanced by certain  substances including 

•

EDTA

•

Butylparaben

•

Tripolyphosphate 

•

other naturally occurring antimicrobial agents

Methods of lysozyme isolation – Direct  crystallisation and precipitation

(1)

Crystallisation with 5% sodium chloride 

(2)

Lysozyme recovery usually ranges from 60‐80% 

(3)

Extremely time consuming and the residual egg white is  too salty for further food application

(4)

Ultrafiltration and diafiltration to desalt the remaining  egg white material after lysozyme separation can reduce 

the salt content and the resultant egg white retains its  foaming ability and foam stability 

Methods of lysozyme isolation

–

direct  membrane filtration

•

Only small amounts of lysozyme (20%) are able to  pass through ultrafiltration membranes. 

•

A 2‐step ultrafiltration process combining the high  flux of the 50 kDa MWCO membrane with the high  selectivity of the 25 kDa MWCO membrane is useful 

for the efficient purification of chicken egg white  lysozyme

Methods of lysozyme isolation ‐

ion‐ exchange chromatography

•

A cation exchange resin can be stirred with egg white until the  bulk of the enzyme has been adsorbed. 

•

Residual egg white is decanted or filtered off and used for  food applications. 

•

Amberlite, CM‐cellulose, CM‐Sephadex and Duolite have been  successfully applied as cation exchangers employed a co‐

polymer of methacrylic acid and divinylbenzene (Duolite C‐ 464) with a rigid macroporous structure for the recovery of  lysozyme. 

•

Freeze‐drying and spray‐drying can be successfully used to  dehydrate liquid lysozyme preparations without loss of 

enzyme activity.

Egg white (mucin-free egg white, 2x dil)

ethanol (30%) , pH 5.8

Centrifugation 4000 rpm, 30 min

Supernatant Precipitate Adjustment pH to 7.4)

adjust to 40% ethanol

Centrifugation Cation exchange chromatography

Conalbumen

Washing Elution

( Phosphate + Nacl) (Phosphate+NaCl)

Lysozyme

Ion Exchange Chromatography (con’d)

Methods of lysozyme isolation ‐

Affinity  chromatography

• Use glucochitin (deaminated chitin) or chitosan (deacylated  chitin)

• Not adapted for large‐scale isolation of lysozyme

‐

high cost of affinity supports or ligands, 

‐

poor flow rates of undiluted egg white, 

‐

expensive and time‐consuming elution procedures 

‐

non‐specific adsorption of other egg white proteins . 

• A dye affinity membrane system produced lysozyme with a  purity of 88% and a recovery of 92%. 

Why lysozyme has been used as an  antimicrobial agent in various foods

•

Heat stable 

•

Active in a broad range of temperatures (from 1 C to nearly  100 C) 

•

Withstands boiling for 1‐2 min 

•

Stable in freeze‐drying and thermal drying 

•

Not inactivated by solvents,

•

Maintains its activity when redissolved in water

•

Has optimum activity at pH 5.3 to 6.4 (i.e. typical for low‐ acidic food) 

•

The presence of other proteins in food, however, can reduce  its stability by the formation of sulfide bridges

Avidin•

Avidin is a trace component (0.05%) of egg white

•

A tetrameric, strongly basic glycoprotein protein•

Composed of subunits of identical amino acid composition and 

sequence (15.6 kDa and 128 amino acids each) •

Combines with biotin

to form a stable complex, which is 

incapable of absorption by the intestinal tracts of animals. •

Avidin binds with 4 biotin molecules.

•

The binding between biotin and avidin is so strong that  separation requires heating the complex at 120 C for 15  minutes. 

•

The high affinity constant of avidin for biotin has been widely  used in molecular biology, affinity chromatography, molecular  recognition and labelling, Enzyme Linked ImmunoSorbent 

Assay (ELISA), histochemistry and cytochemistry

• Other proteins can be separated by this method based on their affinity for specific groups or compounds.

• Examples:

Antigen 

Antibody

Antibody

Antigen

Substrate

Enzyme

Concavalin A

Glycoprotein

Hormone

Binding 

Protein/Receptor

Affinity Chromatography

Lipid and Protein Components of Egg Yolk

Lipids (31%)

Proteins (17%)

Neutral Lipids (65%)

Lipovitellins (α‐

and β‐): 69%Phospholipids (30%)

‐

α‐

Lipovitellins: 58% 

‐

PC (83%)

‐

β‐

Lipovitelins: 11%‐

PE (14%)

Livetins: 12%

‐

Sphingomyelin (2.5%)

‐

α‐

livetin (serum albumin): 4%

‐

Phosphatidylinositol (0.5%)

‐

β‐

livetin (glycoprotein): 5%

Cholesterol (5%)

‐

γ‐

livetin (γ‐globulin): 2%Carotenoids (carotenes)

Phosvitin: 7% 

Xantophylls (lutein, zeaxanthin)  apo Low‐density lipoproteins: 12%

PROTEINS AND LIPOPROTEINS IN YOLK –

Yolk can be separated to granules and plasma by high‐ speed centrifugation 

Plasma

• Make up of 78% of yolk.

• Composed of 49% moisture. 

• On a dry basis, plasma consists of 77‐81% lipid, 2.2% ash, 18%  protein.

• Proteins in plasma are composed of livetin and LDL‐proteins

• Livetins (α‐, β‐, γ‐) are fat‐free globular proteins that  represents 10.6% of the total yolk solids.

• LDL proteins contains 89% lipid which consists of 74% neutral  lipid, 26% PL (75% PC, 18% PE, 8‐9% sphingomyelin and 

lysophospholipids) 

Granules

•

Make up of 22% of yolk.

•

Composed of 44% moisture. 34% lipids, 60% protein, 6% ash  on a moisture‐free basis.

•

Phospholipids (85% PC, 15% PE) makes up 37% of total lipid. 

•

Proteins in granules are composed of 70% α‐

and β‐ lipovitellines, 16% phosvitin, and 12% low‐density lipoprotein.

•

Phosvitin binds ferric iron 

•

Lipovitellines contain about 20% lipids, which include 40%  neutral lipids and 60% PL.

•

LDL contains 84% lipid which consist of 3.7% cholesterol, 31%  phospholipid, and 65% neutral lipid.

Separation of IgY

IgY•

The molecular mass of IgY is ~ 180 kD (light chain ~25 kilo 

Dalton [kD] each: heavy chain ~ 65‐68 [kD] each). 

•

Yolk antibodies are highly resistant to acid and heat–

IgY lost little of antibody activity by incubation for 10 min at

pH 7.2 at 

60

°C or for 10 min at 40

°C above pH 4.0. 

–

Antibody activity were significantly diminished by incubation above  65 °C or below pH 4.0. 

–

30‐50% (w/v) sucrose reduced the heat denaturation of IgY at 75‐ 80 °C 

–

The addition of high levels of sucrose, maltose, glycerol or glycine  displayed effective additional protection against thermal denaturation 

of IgY.

IgY

•

The major serum antibody in chicken is IgG, but IgG  is also transported to egg in a manner similar to the  placental transfer of IgG in mammals. 

•

The protection of relatively immuno‐incompetent  newly hatched chicks from pathogens is through the 

transmission of antibodies from the mother via the  egg. 

•

In the egg, chicken IgG is found mainly in egg yolk,  and the concentration in egg white is very low. 

Methods studied for purification of IgY

Ammonium sulfate precipitation (Schwarzkopf and Thiele 1996)

Dextran sulfate precipitation (Jensenius 1981)

Polyethylene glycol (PEG) precipitation (Polson and Wechmar 1980)

Caprylic acid extraction (McLaren et al. 1994)

Propanol-aceton extraction (Bade and Stegemann 1984)

Affinity chromatography (Chen et al. 2002)

Ion exchange chromatography (Akita and Nakai 1992)

Ultrafiltration (Kim and Nakai 1996, 1998)

Gel filteration (Akita and Nakai 1992)

Other Studies40 ~ 70% loss of IgY by ultrafilteration (Akita and Li-chan1998)

Use of 0.1% carageenan exhibited a 14% loss of IgY from the doubly diluted yolk in natural pH (Chang, 2000)

Akita and Nakai (1992) - pH 4.6-5.2 removed lipids and incubation at cold room for 2 ~6 hrs made WSF clear

- 60% ammonium sulfate precipitation resulted in 89% IgYrecovery with 30% purity

- The best purification scheme was combination of salt precipitation, ultrafilteration, and gel filteration,resulting in over 100 mg of pure IgY production per egg

Ammonium sulfate precipitation (25 ~45%) provided highpurity and 58% recovery of IgY (Svendsen, 1995)

Final Purification steps for IgY

Gel filtration: Devi et al. (2002)

Ammonium sulfate fractionation: Svendsen et al. (1995)

Ion exchange chromatography

‐

Cation exchange (CM‐Cellulose): Fitchtali et al. (1992,  1993) 

‐

Anion exchange (DEAE‐Sephacel): Akita and Nakai (1992)

Affinity chromatography 

‐

Fe3+

affinity: Greene and Holt (1997)

‐

Immunoaffinity chromatography: Chen et al. (2002)

Separation of water soluble proteins  

Dilution of egg yolk‐

10 times cold phosphate buffer, 20 mM  ‐

Adjustment of pH to 5.0 helps lipid removal and

lower ionic strength helps the aggregation of lipoproteins‐

Refrigeration of egg yolk soln. before centrifugation  

Centrifugation ‐

at 2,800 x g, 40 min, 4oC 

‐

water soluble protein extraction 

‐

lipid precipitation

Removal of lipoproteins and Concentration  

After centrifugation, 0.02% carageenan or 0.01% charcoal is added to WSF following filteration with Whatman No. 1 paper Concentration of prepared WSF through ultrafiltration Ultrafiltration

-

Using Labscale TM TFF System with Pellicon XL Biomax 50 membrane filter

-

Cut-off size : 50 kDa

Final step of purification

• Cation exchange chromatography or Ammonium sulfate precipitation for production of high purity IgY

• Dialysis : after ammonium sulfate precipitation removal of remaining salt by dialysis

• ELISA : measuring activity of IgY

• Electrophoresis : optical estimation of IgY purity

Effects of pH adjustment on diluted egg yolk solution

Treatment Lipid (%) Turbidity* Protein (%)

Control 1.0 2.97 0.41

pH to 5.0 0.08 0.33 0.51

* Turbidity: Abs. at 600 nm

Effect of temperature on extraction of  WSF*  by Centrifugation

Treatment Supernatant vol. (%)**

Turbidity (A600 )

IgY (mg/ml)

Total IgY ***(yield x IgY)

Control 77.03a 0.065a 1.11a 86.73a

Refrigeration 78.23a 0.040b 1.12a 88.78a

Freezing 72.03b 0.014c 1.24a 90.05a

* means water soluble proteins obtained by centrifugationafter storage at different temp of diluted egg yolk soln.

** indicates the volume obtained after centrifugation*** After centrifugation and diafilteration. The content of IgY was

measured through the ELISA method

Addition of Carrageenan to WSF 

0.0

0.1

0.2

0.3

0.4

IgY

(mg/

ml)

Control 0.01 0.02 0.05 0.1 0.2 Charcoal0

0.1

0.2

0.3

0.4

IgY

(mg/

ml)

pH 4 pH 5 pH 6 pH 7 pH 8 pH 9

% carrageenan pH condition

The best conditions selected for carageenan to WSF- 0.02% concentration and pH 9.0

Control : without carageenan

Charcoal treatment to WSF isolated  egg yolk

0.0

0.5

1.0

1.5

2.0

2.5

3.0

IgY

(mg/

ml)

4.0 5.0 6.0 7.0 8.0 9.0

pH Condition

0

0.5

1

1.5

2

IgY

(mg/

ml)

0.005 0.01 0.05 0.1

Charcoal %

Best conditions for Charcoal to WSP- pH 4.0 and 0.01% concentration

Conditions of Ammonium sulfate ppt and  Cation exchange chromatography

2.0

3.0

4.0

5.0

6.0

7.0

IgY

(mg/

ml)

4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 C*

pH

0.0

1.0

2.0

3.0

4.0

5.0

6.0

4.0 5.0 6.0 7.0 8.0 9.0

pH

IgY

(mg/

ml)

40% Ammonium sulfateCation exchange chromatography

C* : total IgY amount before loading Washing : 5 Vol. of 20 mM Citrate phosphate

buffer(CB), pH 4.8 Elution : 2 Vol. of 200 mM CB, pH 6.4

Using 40% ammonium sulfate

At pH 9.0 : the best high IgY recovery

Comparison of purity on SDS page gel

M : Marker A : Chicken IgG1 : Centrifugation2 : Charcoal + Diafilteration3 : Ultrafilteration4 : Ammounium sulfate 1st ppt

5 : Ammounium sulfate 2nd ppt

200k

45k

M A 1 2 3 4 5IgY

1 : Loading, at pH 4.8 after Ultrafilteration2~8 : Washing, 20mM CB buffer, pH 5.49 : Elution, 200 mM CB buffer, pH 6.4

Ammonium sulfate precipitaton Cation ion exchange chromatography

1 2 3 4 5 6 7 8 9

Recovery of IgY

Purification step Yield (%)

Water soluble fraction * 100

Charcoal and Diafilteration 103.47 ±

0.03

Ultrafiltration 80.02 ±

13.72

Ammonium sulfate (1st) 74.82 ±

12.57

Ammonium sulfate (2nd) 69.07 ±

7.58

Water soluble fraction of egg yolk was obtained from centrifugation

Conclusion

Developed more simple, efficient, rapid purification method for large‐scale IgY production from egg yolk.

Addition of 0.01%charcoal to water soluble fraction, pH 4.0, resulted in concentrating IgY and reduced attachment of IgY to ultrafiltration membrane.

IgY with 70% recovery and high purity was obtained by 40% ammonium sulfate precipitation at pH 9.0 and 0.01% charcoal addition.

Final preferred method for IgY purification

Liquid egg yolk

10x dilution with phosphate bufferpH adjustment to 5.0 with 1 N-HCl

Centrifugation (2,800 x g, 40 min, 4oC)

0.01% charcoal at pH 4.0 and filteration with Whatman No. 1 paper

Ultrafiltration

40% Ammonium sulfate precipitation at pH 9.0

Dialysis

Phosvitin

•

Phosvitin is a principal phosphoprotein present in egg yolk  (approximately 16% of egg yolk proteins) 

•

Contains ~10% phosphorus. Thus, phosvitin has an excellent  metal (iron and calcium) binding capacity. 

•

The calcium binding properties of phosvitin is influenced by  pH. 

•

The calcium binding capacities of native phosvitin were 20  mol Ca++/mol of phosvitin at pH 3.6 and 148 mol Ca++/mol  phosvitin at pH 7.0 . 

•

Phosvitin was capable of inhibiting lipid oxidation in  phosphotidylcholine liposomes, muscle homogenates, and 

ground pork. 

AEFGTEPDAKTSSSSSSASSTATSSSSSSASSPNRKKPMDEEENDQVKQA

RNKDASSSSRSSKSSNSSKRSSSKSSNSSKRSSSSSSSSSSSSRSSSSSS

SSSSNSKSSSSSSKSSSSSSRSRSSSKSSSSSSSSSSSSSSKSSSSRSSS

SSSKSSSHHSHSHHSGHLNGSSSSSSSSRSVSHHSHEHHSGHLEDDSSSS

SSSSVLSKIWGRHEIYQ

Phosvitin is a principal phosphoprotein present in egg yolk with molecular mass of ~35 kD. Phosvitin contains ~10% phosphorus. O-

⏐O = P – O-

⏐O⏐

Amino Acid Sequence of Egg Yolk Phosvitin

151

101151201

50100150200217

123 phophoserines↓

Metal Chelating CapabilityPhosphorous Source

Use of Phosphopeptides

•

Calcium supplement–

Soluble calcium phosphate formation

–

Inhibiting the formation of insoluble calcium phosphates –

CPP is currently sold as a calcium‐supplementing agent 

and mineral absorption enhancing agent in Japan and  Sweden 

•

Iron supplement–

Soluble iron phosphate formation

•

Antioxidant–

Iron chelating effect

Separation strategies of phosvitin

• Phosvitin is present in yolk granules where electron‐dense lipovitellin  micells are attached to thread‐like phosvitin complexes (Chang et al.,  1977; Radomski and Cook, 1964). 

• The yolk granules are insoluble in hypotonic solution but can be

disrupted  at 1.71M NaCl solution (Chang et al., 1977). 

• Phosvitin can be isolated from egg yolk by precipitating with a magnesium  sulfate (Mecham and Olcott, 1949), and using chromatography (Connelly 

and Tarbosky, 1961)

• Extracting phosvitin from lipid‐free egg yolk powder prepared with  ethanol and hexane combination (1:3, Lesso and Nakai, 1995) 

• Extracting phosvitin from liquid egg yolk with NaCl and then precipitating  it with MgSO4

(Castellani et al., 2003). 

Separation of Lipids, Phospholipids  and Proteins from Egg Yolk

Composition of egg yolk

Sample preparation Moisture Lipids Protein Ash

Whole yolk 48.7 33.2 16.6 1.5

Yolk after water 69.2 20.8 8.9 1.0

extraction (pH 5.0)

Yolk after lipid extraction 10.0 0.8 86.1 3.1

Selection of Solvents for Lipid Extraction from  Yolk

1. Low toxicity: Essential for human consumption

2. Extractability: Lipid and phospholipids

3. Easy to handle

4. Price

5. Recycling

Characteristics of Solvents

• Neutral lipids are soluble in hexane and acetone• Leithin: soluble in ethanol (1:12 vol), insoluble in 

acetone• Cephalins: slightly soluble in ethanol, insoluble in 

acetone• Cholesterol: soluble in ethanol (1.3 g/100 ml), 

soluble in hot ethanol (28 g/100 ml). • Sphinomyelins are freely soluble in hot alcohol.

Ethanol Extraction of Neutral Lipid and  Phospholipids from Egg Yolk

• Ethanol at room temperature first, then 60 C, and  finally at 79 C ethanol (84%, final concentration). 

• 95% of lipid extraction from liquid egg yolk. • Further fractionation using the cold temperature 

crystallization and then filtration.• Yielded > 90% pure lecithin (Sim, 1994).

Extract of Egg Yolk with Acetone and Ethanol

TG

PE

PC

TG PE PCStd

Chol

Actn9 vol.

Actn9 vol.

EtOH 4 vol.

EtOH9 vol.

Ethanol and Hexane Extraction of Neutral  Lipid and Phospholipids

• Water insoluble fraction of diluted egg yolk (67‐69% moisture  content) 

• Mixing with 4 volumes of 100% ethanol yielded 87% and 9  volume produced 93% ethanol (final concentration)

• One ethanol extraction (5 vol.) produced mostly PC and PE,  and little TG.

• Repeated (3‐5 times) extraction with ethanol or higher  ethanol volumes extracted more neutral lipids

• Decrease in ethanol concentration (down to 71%) reduced  the extractability. 

Extraction of Lipids from Egg Yolk

90% ethanol 81% ethanol 72% ethanol 63% ethanol

Further purification of phospholipids and  neutral lipids

• Hexane extraction (4 volumes, 3 times): Mostly neutral lipids and some  phospholipids

• Proteins were collected as the final component of water‐insoluble fraction  of egg yolk. 

• The ethanol and hexane extract of water‐insoluble fraction of egg yolk  were dried using a rotary evaporator. 

• The dried lipids were redissolved

in 85% hot ethanol and then placed in a  water bath to warm up. 

• Lipid layers were separated using a separatory

funnel. 

• The lipid separated from the top was phospholipids ( > 95% purity),  middle layer was neutral lipid ( > 95% purity), and bottom layers were 

phospholipids and neutral lipids mix.

Flow chart for large-scale isolation of lipids and protein from egg yolk

Yolk precipitant (69% moisture)

Extraction of phospholipids with 4 vol. of 100% ethanol(87% ethanol, final concentration)

• Centrifugation at 2,800 x g, 15 min at 4oC 

Supernatant   

Precipitant(Mainly PL) 

(Neutral lipids and protein)

Drying with rotary evaporator                                  

Hexane extraction (4 vol., 3x extraction)(mostly phospholipids) 

(Protein residues are separated)

Drying lipid extracts

redissolving

in ethanol 

Separation of PL and NL

Challenges

• The purity of the bottom layers were not very  high and need further purification. 

• Removal of cholesterol from the phospholipids is  necessary

• Removal of color: pigments such as xantophyll (lutein, zeaxanthine) and carotene.

• Further fraction of phospholipids to PC, PE,  sphingomyelin

and phosphatidylinositol.