Lecture 4

Lecture 4 - Lecture 4 1/14/10 Assignment: Segel, Pages...

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Lecture 4 1/14/10 Assignment: Segel, Pages 141-142: Problems 1, 2, 3 and 5 Background Reading: Segel, Page 94 Outline ± Isoelectric point ± Ion-exchange chromatography ± Amino-terminal amino acid determination ± Carboxyl-terminal amino acid determination ± Specific fragmentation of peptides ± Edman degradation ± Amino acid sequencing ± Functional diversity of proteins ± Primary structure of proteins ± Secondary structure of proteins
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± Isoelectric point The isoelectric point of an amino acid or peptide is the pH at which it carries no net charge. It is often abbreviated as p I H H 2 N H 2 N NH pK a1 = 2.2 pK a2 = 9.0 pK a3 = 12.5 Net charge: +2 +1 0 -1 p I of arginine = pK a2 + pK a3 2 = 10.75
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± Ion-exchange chromatography Cation-exchange using a resin with sulfonic acid groups attached. SO 3 - SO 3 - CH 2 CH CH CH 2 n Dowex-50 SO 3 - This resin would be a cationic – exchange resin. The resin would bind to positively charged molecules. The positive charges of the amino acids will bind to the resin by electrostatic forces. An amino acid with a higher [AA + ]/[AA - ] ratio will elute before one with a lower ratio; in general, an amino acid with a lower p I will elute before one with a higher p I. Hydrophobic R groups of some of the amino acids may also interact with the Nonpolar benzene ring, causing them to bind a little better to the resin.
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Cation-exchange resins are often used in Determining the Amino Acid Composition of a protein. Accomplished by hydrolyzing the peptide or protein with 6 N HCl. This treatment hydrolyzes the peptide bond. The mixture of amino acids is then separated by Cation-exchange Chromatography. Wash column with a buffer at about pH 3 and then apply the mixture of amino acids in this buffer to the column. At this low pH, all of the amino acids will have a positive charge and will bind to the resin. After binding the amino acids to the column, can then elute them
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This note was uploaded on 06/23/2010 for the course BIS 102 taught by Professor Hilt during the Winter '08 term at UC Davis.

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Lecture 4 - Lecture 4 1/14/10 Assignment: Segel, Pages...

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