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Unformatted text preview: BC367 Experiment 1 Identification of an Unknown Amino Acid Introduction As the building blocks of proteins, amino acids play a key cellular role in structure and function. Proteins themselves participate in nearly every physiological event in the cell. In order to understand acid-base properties of proteins and their behavior as polyionic macromolecules, we will begin by investigating the properties of their constituent amino acids. Since all amino acids contain at least one amino and one carboxyl group, they are classified as amphoteric substances (meaning that they can act as either an acid or as a base). Such a molecule reacts with acids as follows: and with bases as follows: The ionic form of the amino acid present in an aqueous solution depends on the pH of the solution. In this experiment, you will identify an unknown amino acid through acid-base titration. Titration curves of amino acids are very useful for identification. As you can see in the example for glycine shown below, a simple amino acid has two dissociation steps corresponding to loss of H + from the acidic carboxyl group at low pH followed by loss of H + from the more basic amino group at high pH. The pK a value for each dissociable group of an amino acid can be determined from such a titration curve by extrapolating the midpoint of each buffering region (the plateau) in the titration curve. The diagram also shows that there is a point in the curve where the amino acid behaves as a "neutral" salt. At this pH, the amino acid is predominantly a zwitterion with a net charge of zero. This point of the titration curve is the “isoelectric point” (pI) and can be approximated as halfway between the two points of strongest buffering capacity (the two pK a values). The isoelectric point (pI) can be estimated by: pI = 1/2 (pK 1 + pK 2 ) where K 1 and K 2 are the dissociation constants of the carboxyl and amino groups, respectively. pK 1 of glycine is 2.35; pK 2 is 9.78. Thus, the pI = 1/2 (2.35 + 9.78) = 6.06, meaning that at pH 6.06, glycine has no net charge. R CH COO- H 3 N + CH COOH H 3 N + K 1 Cationic form Acid Zwitterion + H 2 O + H 3 O + R R CH COO- H 3 N + R CH COO- H 2 N Anionic form + H 2 O Base K 2 + OH- Zwitterion BC 367, Experiment 1, Fall 2009 2 Charged amino acids have acidic or basic side chains (R-groups) giving them more than two dissociable H + ions. For example, glutamic acid has a carboxylic acid side chain in addition to its α-carboxyl and α-amino groups. A titration curve for glutamic acid will be somewhat more complex than that for glycine. Three plateau regions and three pK a values will be observed for glutamic acid: two in the acidic pH region, pK 1 ( α-carboxyl group) = 2.2; pK 2 ( γ-carboxyl group) = 4.3; and one in the basic pH region, pK 3 ( α-amino group)= 9.7. Members of the basic family of amino acids, such as lysine, will also exhibit three pK a values; however, due to the extra amino group they will have one pK a in the acidic pH region and two pK...
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