CHROMATOFOCUSING

by Amlan Barai13I300002Department of Bio-Science & Bio-Engineering Indian Institute of Technology Bombay

pH and Protein

pH=-log[H+]

So, A protein at pH3 have more [H+] in its environment than a protein at pH9

pKa

• pKa is the pH at which any group donates half of its ionisable proton

pKa=-logKa

• pKa tells us how acidic (or not) a given hydrogen atom in a molecule is. The stronger the acid, the lower its pKa; the stronger the base, the higher its pKa

pH

1pH

2.34

Amlan Barai, 13i300002, BSBE, IIT Bombay

pH

6pH

9.69

Amlan Barai, 13i300002, BSBE, IIT Bombay

at pKa1=2.34

Increasing the pH

Amlan Barai, 13i300002, BSBE, IIT Bombay

at pKa2=9.69

Decreasing the pH

Amlan Barai, 13i300002, BSBE, IIT Bombay

The Midpoint : pI or Isoelectric Point

• Now at the Midpoint,i.e at pH = = = 6.015

[COO-] = [NH3+]

Amlan Barai, 13i300002, BSBE, IIT Bombay

So pI or Isoelectric Point

is simply the pH at which the net charge is Zero.

Calculation of pI:the pKa & pI of Amino Acids

Table: Modified from Lehninger Principle of Biochemistry, 5th ed. P-73.

pI of Protein

above pI: -veBelow pI:+ve

So the Bottom line

• Proteins and Amino Acids are Amphoteric molecules with positively and negatively charged groups, where their dissociation depends on the H+ ion concentration of the surrounding environment.• Molecules at pH above its pI have net –ve charge & Below its pI have

net +ve charge.

Chromatofocusing

• Separating on the basis of pI (Isoelectric Point)

• First Discovered in 1978 by Sluyterman And His Colleagues

above pI: -veBelow pI:+ve

Ref: http://macromol.sbcs.qmul.ac.uk/oldsite/expertise/CF3.jpg

The Elution profile of two Proteins

9

8

Ref(modified from): Chromatofocusing- Douglas D Frey,Chittoor R Narahari, Ronald C Bates, Encyclopedia Of Life Sciences /&2001 Nature Publishing Group / www.els.ne

above pI: -veBelow pI:+ve

Focusing in chromato“focusing”

-+++++

++++

+pH<8pH=8(=pI)

pH>8

+-

-

pH<8pH=8(=pI)

pH>8

pH<8pH=8(=pI)

pH>8

+ pH<8pH=8(=pI)

pH>8

+

Elution with G

radually increasing pH

Amlan Barai, 13I300002, BSBE, IIT Bombay.

above pI: -veBelow pI:+ve

Focusing in chromato“focusing”above pI: -veBelow pI:+ve

Chromatofocusing: The Assembly

The pH Gradient

The self generated gradient

5pH5

pH9

Buffers used for chromatofocusing

Polybuffer 74• Polybuffer 74 forms a linear pH gradients from pH 7 to 4.• Use Polybuffer 74 for any pH gradient between 7 and 4.• Developed for chromatofocusing, form linear pH gradients.• Mixtures of selected amphoteric buffering substances of different pI

and pKa values.• Resolves pI differences of 0.04 pH units. Ref:GE Healthcare(http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/products/AlternativeProductStructure_17391/17071201)

& Amersham Pharmacia Biotechnology.

Buffers used for chromatofocusing

Polybuffer 96• Polybuffer 96 forms a linear pH gradients from pH 9 to 6.• Developed for chromatofocusing, form linear pH gradients.• Mixtures of selected amphoteric buffering substances of different pI

and pKa values.• Resolves pI differences of 0.04 pH units.• Use Polybuffer 96 for pH gradients that should begin above pH 7.Ref:GE Healthcare(http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/products/AlternativeProductStructure_17391/17071201) & Amersham Pharmacia Biotechnology.

Beads for chromatofocusing

PBE 94 (Polybuffer exchange94)• PBE 94 is a bead-formed exchanger gel (Sepharose®). Charged groups

has coupled with them via ether linkage.• It has an even capacity over a wide pH range.• It is developed specifically for chromatofocusing with Polybuffer™• Highly stable can even function in presence of 8M Urea and at 120°C.

Ref:GE Healthcare(http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/products/AlternativeProductStructure_17391/17071201) & Amersham Pharmacia Biotechnology.

Application

• Separating proteins according to isoelectric point (pI)• It is a powerful method for high resolution, since it can resolve very

small differences in pI (down to 0.02-0.05 pH units) and thus separate very similar proteins.• Used for analytical separations.

Applied for

• Separation of two isoforms of the proteinb2-macroglobulin that differ by a single amino acid residue(Odani H, Oyama R, Titani K, Ogawa H and Saito A (1990)Biochemical and Biophysical Research Communications)

• Purification and concentration of proteins produced by Haemophilus influenzaefor use in proteome analysis (Fountoulakis M, Langen H, Gray C and Takacs B (1998)Journal of Chromatography A806: 279–291)

• Preparative-scale separation and purification of the peptides thymosinb4 and thymosinb9 from bovine tissue (Roboti A, Livaniou E, Evangelatos GPet al. (1994)Journal of Chromatography A662:27–34)

• Separation of cortisol–bovine serum albumin conjugates (Giraudi G and Baggiani C (1990)Analyst115: 1531–1534)

Reference• Protein Liquid Chromatography-edited by Michael Kastner Chapter7-Chromatofocusing:Richard Lukacin and Wolfgan R. Deppert (http://books.google.co.in/books?id=3WhftkdNpxYC&dq=chromatofocusing+principle)

• Chromatofocusing- Douglas D Frey,Chittoor R Narahari, Ronald C Bates, Encyclopedia Of Life Sciences /&2001 Nature Publishing Group / www.els.ne

• Amersham Pharmacia Biotechnology (1987)Chromatofocusing with Polybuffer and PBE. Uppsala: Amersham.

• Chromatofocusing :L . A . Ae . Sluyterman and J . Wijdenes Isoelectric Focusing On Ion-exchange Columns

Journal of Chromatography, 150 (1978) 31-44Q Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

• GEHealthcare(http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/products/AlternativeProductStructure_17391/17071201)