Determination of Isoelectric Point (pI) By Whole-Column ... ¢§ Isoelectric point for a protein is determined

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  • Determination of Isoelectric Point (pI) By Whole-Column

    Detection cIEF

    Tiemin Huang and Jiaqi Wu

    CONVERGENT BIOSCIENCE

  • Determination of Isoelectric Point (pI)

    by Whole Column Detection cIEF

    Definition q The pH at which the net charge of an amphoteric compound is zero. q Determined by the dissociation of all ionizable functionalities of the amphoteric compound. q A physico-chemical parameter associated with every amphoteric compound. q Affected by parameters such as temperature, media compositions (dielectric constant), and ionic

    strength (which influence the dissociation of ionizable functionality).

    Conclusion q Thus, there is no such thing as an absolute pI value for a given amphoteric compound.

    The pI determination method and conditions must be cited when stating a pI value.

  • Principle of Isoelectric Focusing (IEF)

    When an amphoteric compound is placed in a medium with a pH gradient and subjected to an electric field, it will move towards the electrode with the opposite charge. As it migrates, its net charge and mobility will decrease and it will slow down. Eventually, the amphoteric compound will arrive at the point in the pH gradient where the pH is equal to its pI. Here it will be uncharged and stop migrating.

    At this time, if the amphoteric compound should happen to diffuse to a region outside this pH (in the pH gradient medium), it will pick up a charge and hence move back to the position where it is neutral. In this way amphoteric compounds are condensed, or focused, into sharp stationary bands

    IEF process in an 100 mm ID- 50 mm long capillary

    Sample: 4 human hemoglobin variants 0.5 min

    1 min

    1.5 min

    2 min

    2.5 min

    3.5 min

    4 min

    5 min

    6 min

  • Uniqueness of IEF

    q Highest resolution of all the charge based separation techniques.* q Steady-state separation technique.* q Separation is relatively independent of sample load and the way the sample is introduced*

    * Svensson H. Acta Chem. Scan. 1961, 15, 325-341.

    Righetti P.G. Isoelectric focusing: theory, methodology and application; Elsevier Biomedical

    Press: Amsterdam, 1983

  • pH Gradient Used in IEF

    Natural pH gradient IEF

    q Carrier ampholytes are hundreds or even thousands of amphoteric compounds specially synthesized to have an even distribution of isoelectric points across a given pH ranges.

    q The pH gradient forms naturally by carrier ampholytes under an electric field. q Can be conducted in gel format and capillary format (CIEF). q Resolving power about 0.02 pH unit.

    Immobilized pH gradient IEF

    q Gradient is formed by immobilized pH gradient gel q Highest resolving power about 0.001 pH unit .

  • Slab Gel IEF

    q Commonly conducted on ready made or self making agarose or polyacrylamide gel

    q All steps in a gel IEF analysis are performed manually § Prepare slab gel (if pre-cast gel is used, this

    step is not necessary) § Load samples on the gel § Fix the gel after electrophoresis § Stain the proteins bands separated on the

    gel § Rinse the gel § Analysis of protein band on the gel using an

    imaging system q Suitable for qualitative analysis, and less suitable

    for quantitative analysis. q At present, the most popular IEF format although

    it is labor intensive and time consuming (over hours)

    Example of slab gel IEF Sample : Protein X

    26917-70

    Cathode

    Anode

    1A 2A 3 4 5 6 7 8

    pI markers p

    H g

    ra d

    ie n

    t

  • Capillary Isoelectric Focusing (cIEF)

    q At present, conducted with natural pH gradient IEF (carrier ampholytes-IEF) only q Automation q On-line detection and quantitation q Fast separation and detection (as short as 5 minutes )

    pI marker 5.3

    pI marker 7.4

    Example of cIEF Sample: Protein X pI values and area % are labeled in the e-gram

  • pI Determination

    Standard Methods

    • Computation from all ionizable functionalities of the amphoteric compound. That is basic and acidic amino acids present in the protein

    • Titration

    • cIEF a Measurement of pH after IEF b Calibrated from a mixture of pI markers

  • pI Determination

    1. Algorithm Computation The negative charge (a ) carried by the acidic group of the ampholyte at a given pH can be expressed as

    The positive charge carried by the basic group (a ) of the ampholyte at a given pH can be expressed as

    Ê

    The overall charge (Z) of the ampholytes is

    Ê

    The pI of the ampholyte (the pH where the net charge is zero) can be determined once all the pKa of the ampholytes are known.

    Ê Limitations •Selecting different pK values gives different pI values (i.e., pI of peptide WDDD determined by (1) is 3.38, and it is calculated

    to be 2.82 from the link (2). •The assumption that the ionization of ionizable groups is independent of the others is rarely true. •Modification of proteins is not taken in to account. •The actual folding pattern of the protein is not taken into account (1) Electrophoresis 2000, 21, 603-610 (2) http://www.embl-heidelberg.de/cgi/pi-wrapper.pl

    110

    1 )( +

    = − −

    pHpKa

    110

    1 )( 2 +

    = − +

    pKpH

    =

    =

    + −= n

    i i

    m

    j jZ

    11

  • pI Determination

    2. Titration

    A solution of the amphoteric compound of interest is prepared. The zeta potential over a given pH range is recorded during titration. From the zeta potential (charge) versus pH curve, the isoelectric point (pI) is the pH value of the point that the curve intercepts zero charge. The figure at the right illustrates the titration curve of peptide WDDD.

    Limitation § The isoionic point determined is influenced

    by the buffer ionic strength*. Therefore, the pI based on titration may not be accurate

    * Tanford C. Adv. Protein Chem. 1962,7, 69-165.

    Velick S.F. J. Phys. Colloid Chem. 1949, 53, 135-149

    -5

    -4

    -3

    -2

    -1

    0

    1

    2

    2 3 4 5 6 7 8 9

    pH

    C h a rg

    e

  • pI Determination

    3. cIEF

    a. Measurement of pH after IEF

    § Isoelectric point for a protein is determined by measuring the pH of the protein band or spot on an isoelectric focusing gel

    § The pI determined is accurate when experimental temperature is controlled.

    Ê Limitations § Can only be used for gel IEF, and not

    practical for CIEF at present. § The influence of carbon dioxide to the gel

    system has to be controlled

    Measurement of pH values on IEF gel

    Anodic

    Cathodic

    pH meter With micro probe

  • pI Determination

    b. Calibrated by a Mixture of pI Markers

    § The pI is determined by using a series of calibrated mixture of pI markers

    § Performed on slab gel IEF and cIEF.

    Limitations § Assume the pH gradient is linear. § Assume the given value of pI markers is

    correct

    pI markers bands

    Gel IEF: pI range of the sample is in 7.5 Ð 8.4

    Unknown samples

    Marker Marker

    Marker

    Unknown sample

    7.6 7.7

    7.9

    cIEF

  • Instrument Used for cIEF Method

    Dialysis Hollow Fiber

    IEF Column Inlet CapillaryOutlet

    H+ OH-

    + -

    Detector: Camera in UV

    Light Beam at 280 nm

    Focused Zones

    Sample injection

    iCE280 Analyzer

  • pI Determination Using cIEF Ð

    Ideal Conditions

    q pI markers with accurate pI values

    q Linear pH gradient created by used carrier ampholytes (usually a single carrier ampholyte is used)

  • Ideal Conditions Ð Determining

    Markers’ pI Values

    q 15 synthesized peptides are used as the pI markers (Electrophoresis, 21, 603(2000))

    q pI values of the markers are measured by measuring pH along IEF gel after IEF of the markers

    Measurement of pH values on IEF gel

    Anodic

    Cathodic

    pH meter With micro probe

  • 2

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    0 500 1000 1500 2000

    Peak position (pixel)

    p I v a lu

    e

    Ideal Conditions Ð Determining pH

    Linearity for Carrier Ampholytes

    3

    4

    5

    6

    7

    8

    9

    10

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    Peak position (pixel)

    p I v

    al u

    e

    Ampholine 3.5-9.5

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    Peak position (pixel)

    p I

    v a

    lu e

    Biolyte 3-10

    Servalyt 2-11

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    10

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    Peak position (pixel)

    pI v

    al ue

    Pharmalyte 3-10

  • Ideal Conditions Ð Determining pH

    Linearity for Carrier Ampholytes

    3

    4

    5

    6

    7

    8

    9

    10

    0 500 1000 1500 2000

    Peak position (pixel)

    pI v

    al ue

    r2=0