question answers on amino acid classification calculation of pi
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What is the isoelectric point? Explain a protein purification method based on pI.
Isoelectric pointFrom Wikipedia, the free encyclopedia
The isoelectric point (pI) is the pH at which a particular molecule or surface carries no net electrical charge.Amphoteric molecules called zwitterions contain both positive and negative charges depending on thefunctional groups present in the molecule. They are affected by pH of their surrounding environment and can become more positively or negatively charged due to the loss or gain of protons (H+).The pI value can also affect the solubility of a molecule at a given pH. Such molecules have minimum solubility in water or salt solutions at the pH which corresponds to their pI and often precipitate out of solution. Biological amphoteric molecules such as proteins contain both acidic and basic functional groups. Amino acids which make up proteins may be positive, negative, neutral or polar in nature, and together give a protein its overall charge. At a pH below their pI, proteins carry a net positive charge; above their pI they carry a net negative charge. Proteins can thus be separated according to their isoelectric point (overall charge) on a polyacrylamide gelusing a technique called isoelectric focusing, which utilizes a pH gradient to separate proteins. Isoelectric focusing is also the first step in 2-D gel polyacrylamide gel electrophoresis.
Calculating pI values
For an amino acid with only one amine and one carboxyl group, the pI can be
calculated from the pKa's of this molecule.
For amino acids with more than two ionizable groups, such as lysine, the same formula is used, but this time the two pKa's used are those of the two groups that lose and gain a charge from the neutral form of the amino acid.Lysine has a single carboxylic pKa and two amine pKa values (one of which is on the R-group), so fully protonated lysine has a +2 net charge. To get a neutral charge, we must deprotonate the lysine twice , and therefore use theR-group and amine pKa values (found at List of standard amino acids).
However, a more exact treatment of this requires advanced acid/base knowledge and calculations.The pH of an electrophoretic gel is determined by the buffer used for that gel. If the pH of the buffer is above the pI of the protein being run, the protein will migrate to the positive pole (negative charge is attracted to a positive pole). If the pH of the buffer is below the pI of the protein being run, the protein will migrate to the negative pole of the gel (positive charge is attracted to the
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negative pole). If the protein is run with a buffer pH that is equal to the pI, it will not migrate at all. This is also true for individual amino acids.
What are the different ways in which amino acids can be classified? Illustrate with examples.
Figure 1. A Venn diagram showing the relationship of the 20 naturally occurring amino acids to a selection of physio-chemical properties thought to be important in the determination of protein structure
Classification of Amino Acids
There are twenty amino acids that are used to form proteins in the human body, these are called the proteinogenic2 amino acids. There appear to be many different classification systems, three of which are presented here.
Timberlake3, classifies the amino acids using the system presented in Table 1. She uses a simple method of classification, identifying amino acids as polar or non-polar. A further subclassification
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of acidic-polar when the side chain contains a carboxylic acid, and basic-polar when the side chain contains an amino group is also introduced.
Classification Amino Acid
Nonpolar
Glycine
Alanine
Valine
Leucine
Isoleucine
Proline
Methionine
Phenylalanine
Tryptophan
Polar
Serine
Threonine
Asparagine
Glutamine
Cysteine
Tyrosine
Acidic (Polar)Aspartic Acid
Glutamic Acid
Basic (Polar)
Lysine
Arginine
Histidine
Table 1 Classification of amino acids (after Timberlake3)
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Devlin4 classifies amino acids along structural lines. Devlin’s system is presented in Table 2.
Superstructure Structure Amino Acid
Monoamino, moncarboxylic
Glycine
L-Alanine
Unsubstituted
L-Valine
L-Leucine
L-Isoleucine
HeterocyclicL-Proline
L-Phenylalanine
AromaticL-Tyrosine
L-Tryptophan
Thioether L-Methionine
HydroxyL-Serine
L-Threonine
Mercapto L-Cysteine
CarboxamideL-Asparagine
L-Glutamine
Monamino, dicarboxylic L-Aspartate
L-Glutamate
Diamino, monocarboxylic
L-Lysine
L-Arginine
L-Histidine
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Table 2 Classification of amino acids (after Devlin4)
A third method of classification, sourced from Koolman2 is presented in Table 3. This classification system is again based on structure of the side chain.
Classification Amino Acid
Alphatic (do not contain N,O,S in side chain)
Glycine
Alanine
Valine
Leucine
Isoleucine
Sulfur-containingCysteine
Methionine
Aromatic (benzene ring in side chain)
Phenylalanine
Tyrosine
Tryptophan
Neutral (hydroxyl or amide groups in side chain)
Serine
Threonine
Asparagine
Glutamine
Acidic (carboxylate groups in side chain)
Aspartic acid
Glutamic acid
BasicLysine
Arginine
Imino acid (special case) Proline
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Table 3 Classification of amino acids (after Koolman2)