Digitally signed by Jason Raquin Roque DN: cn=Jason Raquin Roque, o, ou, email=jason_mike15@yahoo. com, c=PH Date: 2012.05.

15 14:21:22 +08'00'

Titration of Amino Acids and Peptides

Roque, Jason R. Hernandez, Ritz Hendrie C. Frias, Abigail Pauline F. Someros, Kristine Carl S. Bachelor of Science in Biology Major in Human Biology College of Science De La Salle University Dasmarias Dasmarias, Cavite, Philippines

ABSTRACT

In this experiment, it will give a deeper understanding about the acid-base behavior of amino acids and peptides. It also aims to identify the unknown amino acid that is assigned to the students through the titration curve. Also, the acid-base behavior of amino acids and peptides will be able to deduce its implication as biological buffers. The experimental method will be a 0.20g sample of the unknown amino acid and aspartame powder. A measure of 25.0 mL of distilled water was added to the sample. To acidify the solution until 1.50, small increments of 0.200 M HCl was added. To be able to get the pKa and pI of the samples, small amount of 0.200 M NaOH was added to the solution and measure its pH until it reaches the 11.5 pH. A titration curve was drawn to illustrate the experimental pKa and pI. Following through the method the unknown amino acid was identified as Aspartic Acid. It is determined also through the comparison of the experimental pKa and pI to the standard table of pKa and pI of the amino acids.

INTRODUCTION

The amino acids (2-aminocarboxylic acids) fulfill various functions in the organism. Above all, they serve as the components of peptides and proteins. Only the 20 proteinogenic amino acids are included in the genetic code and therefore regularly found in proteins. Some of these amino acids undergo further (post-translational) change following their incorporation into proteins. Amino acids or their derivatives are also form components of lipidse. g., serine in phospholipids and glycine in bile salts. Several amino acids function as neurotransmitters themselves, while others are precursors of neurotransmitters, mediators, or hormones. Amino acids are important (and sometimes essential) components of food. Specific amino acids form precursors for other metabolitese. g., for glucose in gluconeogenesis, for purine and pyrimidine bases, for heme, and for other molecules. Several nonproteinogenic amino acids function as intermediates in the synthesis and breakdown of proteinogenic amino acids and in the urea cycle. (2)

Figure 1 Common amino acids have the general structure depicted in Figure 1. They contain in common a central alpha (a)-carbon atom to which a carboxylic acid group, an amino group, and a hydrogen atom

are covalently bonded. In addition, the a-carbon atom is bound to a specific chemical group, designated R and called the side chain that uniquely defines each of the 20 common amino acids. Figure 1 depicts the ionized form of a common amino acid in solution at pH 7. The a-amino group is protonated and in its ammonium ion form; the carboxylic acid group is in its unprotonated or carboxylate ion form. Table 1. Complete Naming and Structure of the Amino Acids (3) Amino Acid Alanine 3-letter code Ala 1-letter code A Properties aliphatic hydrophobic neutral polar hydrophilic charged (+) Structure (un-ionized form)

Arginine

Arg

Asparagine

Asn

polar hydrophilic neutral

Aspartate

Asp

polar hydrophilic charged (-)

Cysteine

Cys

polar hydrophobic neutral

Glutamine

Gln

polar hydrophilic neutral

Glutamate

Glu

polar hydrophilic charged (-)

Glycine

Gly

aliphatic neutral aromatic polar hydrophilic charged (+)

Histidine

His

Isoleucine

Ile

aliphatic hydrophobic neutral

Leucine

Leu

aliphatic hydrophobic neutral

Lysine

Lys

polar hydrophilic charged (+)

Methionine

Met

hydrophobic neutral

Phenylalanine

Phe

aromatic hydrophobic neutral

Proline

Pro

hydrophobic neutral

Serine

Ser

polar hydrophilic neutral

Threonine

Thr

polar hydrophilic neutral

Tryptophan

Trp

aromatic hydrophobic neutral

Tyrosine

Tyr

aromatic polar hydrophobic

Valine

Val

aliphatic hydrophobic neutral

Structures of the common amino acids are shown in Table 1. Alkyl amino acids have alkyl group side chains and include glycine, alanine, valine, leucine, and isoleucine. Glycine has the simplest structure, with R = H. Alanine contains a methyl (CH3) side chain group. Valine has an isopropyl R group (Table 1). The leucine and isoleucine R groups are butyl groups that are structural isomers of each other. In leucine the branching in the isobutyl side chain occurs on the gamma (g)-carbon of the amino acid. In isoleucine it is branched at the beta (b)-carbon. (4)

Figure 2. Alkyl side chains of valine, leucine, and isoleucine. The aromatic amino acids are phenylalanine, tyrosine, and tryptophan. The phenylalanine R group contains a benzene ring, tyrosine contains a phenol group, and the tryptophan R group contains the heterocyclic structure, indole. (4)

Figure 3. Side chains of aspartate and glutamate. In each case the aromatic moiety is attached to the a-carbon through a methylene (CH2) carbon (Table 1). Sulfur-containing common amino acids are cysteine and methionine. The cysteine side chain group is a thiolmethyl (HSCH2). In methionine the side chain is a methyl ethyl thiol ether (CH3SCH2CH2). There are two hydroxy (alcohol)-containing common amino acids, serine and threonine. The serine side chain is a hydroxymethyl (HOCH2). In threonine an ethanol structure is connected to the a-carbon through the carbon containing the hydroxyl substituent, resulting in a secondary alcohol structure (CH3CHOHCHa). (4)

Figure 4. Guanidinium and imidazolium groups of arginine and histidine.

The proline side chain is unique in that it incorporates the a-amino group. Thus proline is more accurately classified as an a-imino acid, since its a-amine is a secondary amine with its a-nitrogen having two covalent bonds to carbon (to the a-carbon and side chain carbon), rather than a primary amine. Incorporation of the a-amino nitrogen into a five-membered ring constrains the rotational freedom around the NaCa bond in proline to a specific rotational angle, which limits participation of proline in polypeptide chain conformations. (4) 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. (5) 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 / REAGENTS & EXPERIMENTAL PROCEDURE

To obtain the necessary result according to the objective given experimental method was used by the students. The students weighed and approximate 0.20g sample of the unknown amino acid and Aspartame powder through the use of analytical balance. The samples were quantitatively transferred to a 250 mL Erlenmeyer Flask. 25.0 mL of distilled water was added to the samples using a volumetric pipette and dissolve the samples by swirling. In order to acidify the solutions up to the pH of 1.5, small increments of 0.200 M HCl was little by little added to the solution. In every addition of HCl, the students measured its pH and record it. When the solution has already reached the pH of 1.5, basifying the solution will be the next step. Small amount of 0.200 M NaOH was also, little by little added to the solution until it reaches 11.5 of pH. Using the data gathered in the acidifying and basifying the solution, a titration curve was drawn. The titration curve will show the pKa and pI of the unknown amino acid and Aspartame. After, getting the pKa and pI, it was compared to the standard table of pKa and pI of the amino acids to determine the identity of the amino acid.

DATA & RESULTS

Through the materials and methods that were used in this experiment, the identification of unknown amino acid will be determined. Titration of the unknown amino acid will also help to identify it. The table below shows the change in pH as the Volume of NaOH increases.

Table 2. Aspartic Acid Vol. NaOH(ml) 0.2 0.4 0.6 0.8 1.2 1.6 2.0 2.4 3.0 pH 1.58 1.60 1.62 1.67 1.72 1.75 1.77 1.79 1.82 Vol. NaOH(ml) 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 pH 2.74 3.08 3.33 3.59 3.60 3.99 4.22 4.53 6.28

3.6 4.2 4.8 5.6 6.4 7.2

1.97 2.03 2.14 2.20 2.51 2.56

17.0 18.0 19.0 20.0 21.0 22.0

7.36 8.47 8.88 9.15 9.64 11.5

Table 3. pKa Values, pI & Titrimetric profile Experimental 2.03 3.99 9.64 3.01 Theoretical 2.09 3.86 9.82 2.98 % Error 3.00% 3.37% 1.83% 1.01% Aspartic acid

pkA1 pKa1 pKa1 pI

Identity of unknown Amino Acid

Graph 1. Titration Curve of Unknown Amino Acid

9.64

pI=3.01 2.03

3.99

As the results above have been illustrated, it is concluded that the unknown amino acid is Aspartic Acid. It was determine by comparing the experimental pKa with the standard pKa of the amino acids.

Aspartame is an artificial sweetener. Although it has roughly the same number of calories per gram as table sugar (sucrose), it is around 200 times sweeter. It is also known by the brand names, Nutrasweet, Equal, Spoonful and Equal Measure (1). Aspartic acid and phenylalanine are the amino acids that bonded to produce an aspartame. (6) The process is illustrated below

Table 4. Vol. NaOH(ml) 1.0 2.0 3.0 4.0 4.5 5.0 5.5 6.0 6.5 7.0

Aspartame

pH 1.68 2.03 4.25 6.12 8.56 9.08 9.63 10.35 10.95 11.5

Table 5. pKa Values, pI & Titrimetric profile Experimental 2.03 4.25 9.63 3.14 Theoretical 1.83 3.86 9.82 2.85 % Error 10.93% 10.10% 12.12% 10.18%

pkA1 pKa1 pKa1 pI

Graph 2. Titration of Aspartame

pKa=9.63

pI=3.14

pKa=4.25 pKa=2.03

Today, aspartame has established itself as an important component in thousands of foods and beverages. It is found in about 6,000 products around the world, including carbonated soft drinks, powdered soft drinks, chewing gum, confections, gelatins, dessert mixes, puddings and fillings, frozen desserts, yogurt, tabletop sweeteners, and some pharmaceuticals such as vitamins and sugar-free cough drops. Consumer research shows that low- and reduced-calorie foods and beverages have become part of the lifestyle of millions of men and women who want to stay in better overall health, control their weight, or simply enjoy the many low- or reduced-calorie products available. Aspartame has helped provide calorie-conscious consumers with a wide variety of good-tasting, low- and reduced-calorie products that are easily incorporated into a healthful lifestyle. Its excellent taste and suitability for a wide variety of products make it an appropriate choice for people who desire a sweet taste without all the calories of sugar. Currently, aspartame is found in more than 6,000 products and is consumed by over 200 million people around the world. (7) Some health benefits from the consumption of aspartame is that 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 With these benefits, aspartames popularity rapidly increase. But still it has a side effects like; 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 inthe blood, blocking or lowering the levels of serotonin, tyrosine, dopamine, norepinephrine, andadrenaline. Therefore, it is typical that aspartame symptoms cannot be detected in lab tests andon x-rays. Textbook disorders and diseases may actually be a toxic load as a result of aspartamepoisoning (7)

REFERENCE (1) (2) (3) (4) (5) Legaspi, G. A. 2009. Essentials of Biochemistry Laboratory Koolman. 2005. Color Atlas of Biochemistry, 2nd edition. Thieme 2005 Retrieved from http://wbiomed.curtin.edu.au/biochem/tutorials/AAs/AA.html on July 6, 2011 Devlin, T. M. Textbook of biochemistry: with clinical correlations, 4th edition. McKee, T. McKee, J.R. 2003. Biochemistry-The Molecular Basis of Life. 3rd Edition.McGraw-Hill Companies, Inc (6) Retrieved from http://www.3dchem.com/molecules.asp?ID=24 on July 6, 2011 (7) Retrieved from http://www.caloriecontrol.org/sweeteners-and-lite/sugar-substitutes/aspartame on July 6, 2011