Titration of Amino acids and peptides

Sharmane Jarin De La Salle University-Dasmarias Dasmarias, Cavite

ABSTRACT The acid-base behavior of amino acids and peptides were analyzed with the use of titration. Titrimetric profile reveals which species predominates at a particular pH. It is also useful in determining the isoelectric pH of the sample.(1) A 0.20 g sample of unknown amino acid powder and aspartame were each added with HCl then titrated with the addition of 0.200M NaOH. The pH of the acidified solution was measured from 1.5 to 11.3 and it was recorded after every addition of the base. The total volume of the unknown sample was 39.2 ml and aspartame has a 30.8ml. A titration curves was constructed to determine the pKas to identify the unknown sample. The unknown sample was lysine. The titrimetric profile of lysine and aspartame were drawn. The pI value for aspartame was 2.7 with 5.26 % error and the pI for lysine was 9.55 with 3.24% error. The experimental and theoretical showed significant differences due to INTRODUCTION Protein is a large molecule composed of one or more chains of amino acids in a specific order determined by the base sequence of nucleotides in the DNA coding for the protein. Proteins are required for the structure, function, and regulation of the body's cells, tissues, and organs. Each protein has unique functions. Amino acid is an organic compound containing an amino group (NH2) and a carboxyl group (-COOH). Since amino acids contain both an acidic and a basic group, they undergo an intramolecular acid-base reaction and exist primarily in the form of a dipolar ion, or a zwitterion.(4) Peptide molecules are composed of two or more amino acids joined through amide formation involving the carboxyl group of each amino acid and the amino group of the next. The chemical bond between the carbon and nitrogen atoms of each amide group is called a peptide bond.(5) Titration is a useful tool in determining the reactivity of amino acid side chains. Because amino acids contain an ionizable group, the predominant ionic form of these molecules in solution depends on pH. Titration of amino acid illustrates the effect of pH on amino acid structure.(3) It is also useful in determining the isoelectric pH of the sample. While the titration curve reveals the pKa of the various prototropic groups in amino acids and peptides and their respective pI values.(1) MATERIALS AND METHODS Exactly 0.20g sample of the unknown amino acid powder was weighed using a analytical balance and was placed on a separate 250-ml Erlenmeyer flask. A 25ml of distilled water was added to each flask using a volumetric pipette then the sample was dissolved by swirling. Small increments of 0.200 M HCl was added and dissolved using a burette afterwards the mixture was swirled and the pH was measured. The procedure was repeated until pH of 1.50 was reached. The acidified solution with 0.200M NaOH was titrated by adding small increments of the base. The flask was swirled and the pH was measured. The pH value at each amount of base added was recorded until pH of 11.50 was reached. The procedures was repeated using commercial aspartame as the new sample . RESULTS AND DISCUSSIONS

The Identity of the unknown amino acid sample was identified as lysine based on its pKa and pI value. It was based on the closest pKa of amino acid in the table of pKa values for protropic groups in amino acid. The added base on the unknown sample until it reached pH 11.3 was 39.2 ml. A. Titration Curve of the Unknown Amino Acid

Experimental Theoretical pKa1 2.4 2.18 pKa2 8.4 8.95 pKa3 10.7 10.79 pI 9.55 9.87 Identity of Unknown Amino Acid: lysine B. Identity of Unknown Amino Acid

%Error 10.09 6.15 0.83 3.24

From adding NaOH, the pH of the acidic solution rises slowly and it determines that the acidic sample resist the change in pH. Based also on the pI of the amino acid, it can be a buffer on physiologic or basic pH. The volume of NaOH was too high before it reached the pH of 11.3 because the pH meter didnt work well so we added too many HCl. The pK1 was 2.4 , pK2 was 8.4 and pK3 was 10.7. the buffering region was the zwitterions. During titration the carboxylate, -CO2-, is the one that acts as the basic site and accepts a proton in acid solution, and its the ammonium cation, -NH3+, that acts as the acidic site and donates a proton in a base solution. In a strongly acidic solution it is present mainly in the form in which the

carboxyl group is uncharged. Under this circumstance the molecules net charge is +1, because the ammonium group is protonated. Lowering the H+ concentration results in the carboxyl group losing its proton to become a negatively charged carboxylate group. At this point, there is no net charge and is electrically neutral. The pH at which this occurs is called the isoelectric point (pI). Because there is no net charge at the isoelectric point, amino acids are least soluble at this pH. (3) As the titration continues, the ammonium group loses its proton and the molecule then has a net negative charge. The isoelectric point is when the amino acid is zwitterionic. Aspartame or Aspartylphenylalanine dipeptide, is a low-calorie sweetener which is which is 200 times sweeter than sucrose. Aspartic acid and phenylalanine are the amino acids that bonded to produce a aspartame.(4) The total volume of NaoH added until pH of the Aspartame reached 11.3 was 30.8ml. A. Titration Curve of Aspartame

B. pKa Values and pI of Aspartame

pKa1 pKa2 pKa3 pI Experimental 2.00 3.4 9.8 2.7 Theoretical 1.83 3.86 9.82 2.85 %Error 9.29 11.92 .20 5.26

Based on the percent error on the pKa and pI values of the unknown sample and the aspartame, there were significant differences in the theoretical and experimental values. Some possible source of errors on the experiment was the inaccurate plotting of pKa values on the titration

curve. If the pKa was wrong, the identity of the unknown sample will be incorrect. The buffering zone of lysine was between the pK1 and pK2 and between pK2 and pK3 .which is 9.55 and the aspartame has a 2.7. Lysine is a basic amino acid, It contains the classic -COOH group and an amino group stemming off of the chiral center for the molecule. It has a basic side chain with a terminal amino group. In aqueous solutions (i.e. the cell) it acts as a base. The rapid rise in aspartames popularity can be attributed to the many benefits aspartame provides to the many calorie-conscious consumers. It has a taste very similar to sugar, enhances the flavors, it does not promote tooth decay, there is a scientific studies show that aspartame is beneficial in weight control and it is helpful for individuals with diabetes that allow them to satisfy their taste for sweets without affecting blood sugar which helps them to comply with a healthful meal plan. In addition, consuming products with aspartame can result in fewer calories, which helps people with diabetes, manage their weight. Under increased temperature, such as baking technique, aspartame hydrolyzes into its respective amino acids. Food products with a higher pH, result in the degeneration of this sweetener. In beverages that are marketed in a powdered form, the aspartame undergoes non-enzymatic browning reaction, referred to as Maillard reaction, thereby resulting in disappearance of flavor and aroma. When consumed within the recommended intake, it is also advised for pregnant mothers. It substitutes their craving for sweets, thereby keeps a check on the calories.(4) But adverse side effects of aspartame consumption includes loss of memory, seizures, headache, blindness, protruding eyes, ringing sound in the ear, palpitation, depression, lack of sleep, breathlessness, diarrhea, and skin rashes. Aspartame changes the ratio of amino acids in the blood, blocking or lowering the levels of serotonin, tyrosine, dopamine, norepinephrine, and adrenaline. Therefore, it is typical that aspartame symptoms cannot be detected in lab tests and on x-rays. Textbook disorders and diseases may actually be a toxic load as a result of aspartame poisoning.

REFERENCES

(1) Legaspi, G.A. 2011. Essentials of Biochemistry Laboratory Handouts (2) McKee, T. McKee, J.R. 2003. Biochemistry-The Molecular Basis of Life. 3rd Edition. (3) McMurry, J. Simanek E. 2008. Fundamentals of Organic Chemistry. 6th edition. The (4) Retrieved (5)
McGraw-Hill Companies, Inc Thompson Corporation 7 July 2010 from http://www.diethealthclub.com/articles/24/diet-andwellness/the-truth-about-aspartame.html DONALD VOET and JUDITH G. VOET, Biochemistry, 3rd ed. (2004). Retrieved 10 July 2011 from http://www.britannica.com/EBchecked/topic/450900/peptide