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Question Answers on amino acid classification calculation of pI

Question Answers on amino acid classification calculation of pI

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Published by: Aditi Patil on Nov 25, 2008
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What is the isoelectric point? Explain a protein purificationmethod based on pI.
Isoelectric point
From Wikipedia, the free encyclopedia
isoelectric point
(pI) is thepHat which a particularmoleculeor surface carries no netelectrical charge.Amphotericmolecules calledzwitterionscontain both positive and negative charges depending onthefunctional groupspresent in the molecule. They are affected by pH of theirsurrounding environment and can become more positively or negatively chargeddue to the loss or gain of protons(H
). The pI value can also affect the solubility of a molecule at a given pH. Suchmolecules have minimumsolubilityin water or salt solutions at the pH whichcorresponds to their
and oftenprecipitateout osolution. Biological amphoteric molecules such asproteinscontain both acidic and basicfunctional  groups. Amino acids which make up proteins may be positive, negative, neutralor polar in nature, and together give a protein its overall charge. At apHbelowtheir pI, proteins carry a net positive charge; above their pI they carry a netnegative charge. Proteins can thus be separated according to their isoelectricpoint (overall charge) on apolyacrylamide gelusing a technique calledisoelectric  focusing, which utilizes a pH gradient to separate proteins. Isoelectric focusing isalso the first step in2-D gel polyacrylamide gel electrophoresis.
Calculating pI values
For anamino acidwith only oneamineand onecarboxylgroup, the pI can be calculated from thepKa's of this molecule.For amino acids with more than two ionizable groups, such aslysine, thesame formula is used, but this time the two pKa's used are those of the twogroups that lose and gain a charge from the neutral form of the aminoacid.Lysinehas a single carboxylic pKa and two amine pKa values (one of whichis on theR-group), so fully protonated lysine has a +2 net charge. To get aneutral charge, we must deprotonate the lysine twice , and therefore use theR-groupand amine pKa values (found atList of standard amino acids). However, a more exact treatment of this requiresadvancedacid/baseknowledge and calculations.  ThepHof an electrophoretic gel is determined by thebufferused for that gel. If  thepHof the buffer is above the pI of the protein being run, theproteinwill migrate to the positive pole (negative charge is attracted to a positive pole). If thepHof the buffer is below the pI of theproteinbeing run, theproteinwill migrate to the negative pole of the gel (positive charge is attracted to the
negative pole). If theproteinis run with a buffer pH that is equal to the pI, it willnot migrate at all. This is also true for individual amino acids.
What are the different ways in which amino acids can be classified? Illustratewith examples.
Figure 1. A Venn diagram showing the relationship of the 20 naturally occurring amino acids to aselection of physio-chemical properties thought to be important in the determination of proteinstructure
Classification of Amino Acids
There are twenty amino acids that are used to form proteins in the human body, these are calledthe
amino acids. There appear to be many different classification systems, threeof which are presented here.Timberlake
, classifies the amino acids using the system presented in Table 1. She uses a simplemethod of classification, identifying amino acids as polar or non-polar. A further subclassification
of acidic-polar when the side chain contains a carboxylic acid, and basic-polar when the sidechain contains an amino group is also introduced.
ClassificationAmino Acid
NonpolarGlycineAlanineValineLeucineIsoleucineProlineMethioninePhenylalanineTryptophanPolarSerineThreonineAsparagineGlutamineCysteineTyrosineAcidic (Polar)Aspartic AcidGlutamic AcidBasic (Polar)LysineArginineHistidine

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