ISOELECTRIC

Isoelectric calculates the isoelectric point of a protein from its amino acid composition assuming no electrostatic interactions occur that perturb ionization. Isoelectric makes a plot of the total positive and negative charges and the net charge of a protein as a function of pH. The isoelectric point (pH at which the net charge is zero) is indicated on the plot. If you use -OUTfile, you can also obtain an output file with a listing of the data.

EXAMPLE

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Here is a session using Isoelectric to determine the isoelectric point of human adenylate kinase (PIR:Kihua):

% isoelectric -OUTfile=kihua.iso

  ISOELECTRIC of what protein sequence ?  PIR:Kihua

                  Begin (* 1 *) ?

                End (*   194 *) ?

  When your LaserWriter attached to tty07 is ready, press <Return>.

OUTPUT

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Here is part of the output file (if you are reading the Program Manual, you can see the plot from this session following the output file):

 ISOELECTRIC of: pir1:Kihua Check: 1665 from: 1 to: 194  August 29, 1998 16:44

P1;KIHUA - adenylate kinase (EC 2.7.4.3) 1 - human

N;Alternate names: myokinase

C;Species: Homo sapiens (man)

C;Date: 23-Oct-1981 #sequence_revision 23-Oct-1981 #text_change 05-Sep-1997

C;Accession: A33508; A00679

R;Matsuura, S.; Igarashi, M.; Tanizawa, Y.; Yamada, M.; Kishi, F.; Kajii, T.;

 Fujii, H.; Miwa, S.; Sakurai, M.; Nakazawa, A

Amino Acid         Number of

                   Residues

-----------------  -----------

Arginine              13

Lysine                19

Histidine              2

Tyrosine               7

Cysteine               2

Glutamic Acid         20

Aspartic Acid          9

Amino Terminus         1

Carboxyl Terminus      1

                       Number of Hydrogen Ions Bound

         ----------------------------------------------------------    Net

  pH     Arg   Lys   His   Tyr   Cys   Glu   Asp   NH2  COOH  Total   Charge ..

  1.0   13.0  19.0   2.0   7.0   2.0  20.0   9.0   1.0   1.0   74.0    34.97

  1.5   13.0  19.0   2.0   7.0   2.0  20.0   9.0   1.0   1.0   73.9    34.92

 ///////////////////////////////////////////////////////////////////////////

 12.5    6.5   0.4   0.0   0.2   0.0   0.0   0.0   0.0   0.0    7.1   -31.94

 13.0    3.1   0.1   0.0   0.1   0.0   0.0   0.0   0.0   0.0    3.3   -35.70

                           Isoelectric Point

  9.1   13.0  18.6   0.0   6.9   0.3   0.0   0.0   0.2   0.0   39.0     0.00

INPUT FILES

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Isoelectric accepts a single protein sequence as input. If Isoelectric rejects your protein sequence, turn to Appendix VI to see how to change or set the type of a sequence.

RELATED PROGRAMS

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PeptideSort shows the peptide fragments from a digest of an amino acid sequence. It sorts the peptides by position, putative molecular weight, and relative HPLC retention at pH 2.1, and shows the composition of each peptide. It also prints a summary of the composition of the whole protein. PeptideSort lists the isoelectric points of the peptide fragments resulting from the digest of an amino acid sequence as well as the isoelectric point of the entire protein sequence.

RESTRICTIONS

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The isoelectric point must exist and lie between pH 1.0 and 13.0.

ALGORITHM

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The isoelectric point is the pH at which the protein has no net charge. The net charge of a protein is calculated as the sum of the number of positively charged residues (protonated lysine, arginine, histidine), minus the number of negatively charged residues (deprotonated tyrosine, cysteine, glutamate, aspartate), plus the number of protonated amino termini, minus the number of deprotonated carboxyl termini. The net charge calculation does not take into account any electrostatic interactions within the protein that may perturb ionization. For each amino acid of interest, the number of protonated residues is determined by the following equation:

N_(p) = N_(t) [H⁽⁺⁾] / ([H⁽⁺⁾] + K_(N))

where N_(p) = number of protonated residues, N_(t) = total number of residues of a specific amino acid, [H⁽⁺⁾] = hydrogen ion concentration, K_(N) = dissociation constant for the amino acid of interest that is equal to the following:

₍₁₀₎ ^(-pK)N

SUGGESTIONS

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By default, the vertical axis of the plot (Net Charge) is scaled from -50 to +50. For some proteins, particularly large ones, the charge may be much less than -50 or much greater than +50 at pH's close to the isoelectric point. For those proteins, you can expand the vertical scale with -MINCharge and -MAXCharge.

GRAPHICS

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Accelrys GCG (GCG) must be configured for graphics before you run any program with graphics output! If the%setplot command is available in your installation, this is the easiest way to establish your graphics configuration, but you can also use commands like % postscript that correspond to the graphics languages GCG supports. See Section 5, Using Graphics in the User's Guide for more information about configuring your process for graphics.

<CTRL>C

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If you need to stop this program, use <Ctrl>C to reset your terminal and session as gracefully as possible. Searches and comparisons write out the results from the part of the search that is complete when you use <Ctrl>C. The graphics device should stop plotting the current page and start plotting the next page. If the current page is the last page, plotters should put the pen away and graphic terminals should return to interactive mode.

COMMAND-LINE SUMMARY

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All parameters for this program may be added to the command line. Use -CHEck to view the summary below and to specify parameters before the program executes. In the summary below, the capitalized letters in the parameter names are the letters that you must type in order to use the parameter. Square brackets ([ and ]) enclose parameter values that are optional.

Minimal Syntax: % isoelectric [-INfile=]pir:kihua -Default

Prompted Parameters:

-BEGin=1 -END=194      sets the range of interest

Local Data Files:

-DATa=isoelectric.dat  assigns file of pK data for amino acids

Optional Parameters:

-OUTfile[=kihua.iso]   writes the number of hydrogen ions bound and net

                         charge at pH 1-13 to an output file

-AMINotermini=1        sets the number of amino terminii on protein

-CARBoxyltermini=1     sets the number of carboxyl terminii on protein

-PHDelta=0.5           calculates number of hydrogen ions bound and net

                         charge every PHDelta pH units in output file

-MINCharge=-50         sets the minimum net charge to display on the plot

-MAXCharge=50          sets the maximum net charge to display on the plot

-NOPLOt                suppresses the plot

All GCG graphics programs accept these and other switches. See the Using

Graphics section of the USERS GUIDE for descriptions.

-FIGure[=filename]  stores plot in a file for later input to FIGURE

-FONT=3             draws all text on the plot using font 3

-COLor=1            draws entire plot with pen in stall 1

-SCAle=1.2          enlarges the plot by 20 percent (zoom in)

-XPAN=10.0          moves plot to the right 10 platen units (pan right)

-YPAN=10.0          moves plot up 10 platen units (pan up)

-PORtrait           rotates plot 90 degrees

LOCAL DATA FILES

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The files described below supply auxiliary data to this program. The program automatically reads them from a public data directory unless you either 1) have a data file with exactly the same name in your current working directory; or 2) name a file on the command line with an expression like -DATa1=myfile.dat. For more information see Section 4, Using Data Files in the User's Guide.

This program reads the public or local version of isoelectric.dat to obtain the pK values for the relevant amino acids. The amino acids listed in the public file are the only ones recognized by the program.

PARAMETER REFERENCE

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You can set the parameters listed below from the command line.

-OUTfile=kihua.iso

Causes Isoelectric to write an output file containing the number of hydrogen ions bound and net charge at each pH point.

-AMINotermini=1

Sets the number of amino termini on the protein.

-CARBoxyltermini=1

Sets the number of carboxyl termini on the protein.

-PHDelta=0.5

Sets the pH increment at which number of hydrogen ions bound and net charge are calculated.

-MINCharge=-50

Sets the minimum value on the charge (vertical) scale.

-MAXCharge=50

Sets the maximum value on the charge (vertical) scale.

-NOPLOt

Suppresses the plot.

The parameters below apply to all GCG graphics programs. These and many others are described in detail in Section 5, Using Graphics of the User's Guide.

-FIGure=programname.figure

Writes the plot as a text file of plotting instructions suitable for input to the Figure program instead of sending it to the device specified in your graphics configuration.

-FONT=3

Draws all text characters on the plot using Font 3 (see Appendix I).

-COLor=1

Draws the entire plot with the pen in stall 1.

The parameters below let you expand or reduce the plot (zoom), move it in either direction (pan), or rotate it 90 degrees (rotate).

-SCAle=1.2

Expands the plot by 20 percent by resetting the scaling factor (normally 1.0) to 1.2 (zoom in). You can expand the axes independently with -XSCAle and -YSCAle. Numbers less than 1.0 contract the plot (zoom out).

-XPAN=30.0

Moves the plot to the right by 30 platen units (pan right).

-YPAN=30.0

Moves the plot up by 30 platen units (pan up).

-PORtrait

Rotates the plot 90 degrees. Usually, plots are displayed with the horizontal axis longer than the vertical (landscape). Note that plots are reduced or enlarged, depending on the platen size, to fill the page.

-NOCLIpping

If the data points on a line fall outside of the window in which the data are supposed to be represented, most programs will clip the graph at the edge of the window. This switch disables that clipping.

Printed: May 27, 2005 12:50

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