Content : Properties of amino acids.. Separation and Analysis of Amino Acids

Transcription

1 قسم الكيمياء الحيوية.دولت على سالمه د. استاذ الكيمياء الحيوية ٢٠١٥-٢٠١٤ المحاضرة الثانية 1

2 Content : Properties of amino acids.. Separation and Analysis of Amino Acids 2

3 3

4 Physical Properties of Amino acids 1.Solubility: All amino acids are soluble in water due to the presence of amino, carboxyl and other hydrophilic groups. 2.Optical activity: Due to the presence of asymmetric carbon atoms all amino acids expect for glycine are optically active. All amino acids found in proteins are of the L-configuration. 4

5 3. Melting points : Amino acids generally melt at higher temparatures, often above 200ºC C. 4.Taste :Amino acids may be sweet( Gly, Ala, Val ), tasteless ( Leu ) or bitter ( Arg, Ile ). Monosodium glutamate is a salt of glutamic acid. It is employed as a flavoring agent in food industry to increase taste and flavor 5

6 5.ACIDIC AND BASIC PROPERTIES OF AMINO ACIDS The general properties of amino acids are: They can act as acids (due to the carboxylic acid group) and bases (due to the amine group), and thus they are excellent buffers They migrate when an electric field is applied Amino acids exist as highly polar ions that are called zwitterions ( double-ions ) Zwitterions are ions that have both a negative and a positive charge within the same structure 6

7 Isoelectric Point Each amino acid has an isoelectric point, (pi) numerically equal to the ph at which the zwitterion concentration ti is at a maximum. The amino acid has no NET charge at its pi; it has one positive and one negative charge. At a ph less than the value of the isoelectric point, the amino acid is protonated and has a POSITIVE charge; at a ph greater than the pi the amino acid is deprotonated and has a NEGATIVE charge. g H 3 N 3 O C R ph < pi OH H H 3 N 3 O C R O ph = pi OH H 2 N Cation Neutral Anion 2 O C R O ph > pi (zwitterion form) 7

8 If the zwitterion is treated with acid, that is, a solution containing H +, the H + will add to the COO - to form COOH. Similarly, il l treating ti the zwitterion i with base will result in the loss of the removable proton attached to the NH + 3 group to form NH 2. The ph at which amino acids have no net charge is called the Isoelectric Point or the pi. 8

9 Amino Acids Above and Below Their Isoelectric Points 9

10 Henderson-Hasselbalch We have calculated the ratio of acid to conjugate base for an -carboxyl group and an -amino group at ph 7.0 We can do this for any weak acid and its conjugate base at any ph using the Henderson-Hasselbalch equation ph= pka+ log [unprotonated form (conjugate base)] [protonated form (weak acid) ] H-H equation can be used to calculate the average charge on an ionizable group at any ph 10

11 Let s examine the behavior of a Weak kacid( id(ha) HA), in aqueous solution. Rl Relates three terms: ph, pk a, and [A ]/[HA]. If you know two of these values, you can determine the third. ph = pk a + log([a ]/[HA]) When [A ] = [HA]: ph = pk a + log(1) ph = pk a + 0 ph = pk a pk a is the ph at which a functional group exists 50% in its protonated form (HA) and 50% in its deprotonated t df form (A ). 11

12 pi Depends on Side Chain The 15 amino acids thiol, hydroxyl groups or pure hydrocarbon side chains have pi = 5.0 to 6.5 (average of the pk a s) Glu and Asp have acidic side chains and a lower PI His, Arg, Lys have basic side chains and higher pi 12

13 2.Write out structures for sequential deprotonation and place pka values over the equilibrium i arrows. Fully protonated 1st proton removed 2nd proton removed Net charge = +1 Net charge = 0 Net charge = -1 So, from looking at the net charges, at different e ph s, amino acids can have different charges! Very important for protein structure!! 13

14 Q:Write The charges of the lysine at different ph At ph=1 the lysine possesses two positive, at ph=5.6 two positive and one negative, at ph=9.7 one positive and one negative and at ph=11 one negative The lysine has an amino group on its side chain, its isoelectric point is at ph=

15 Aspartate (D, Asp): Asp has 3 titratable protons Amino acids can be separated on the basis of their charges at a certain ph 1. pka s for the three groups (look at Table23.2) 2. Draw the structures from fully protonated to fully deprotonated Note that t all amino acids are at one point, electrically ll neutral at some ph value. This ph = isoelectric point (pi) pkas: 2, 3.9, 10 (from Table 3.2) pi = (2+3.9)/2 =

16 A buffer is a solution that resists change in ph following the addition of an acid or base. A buffer can be created by mixing a weak acid (HA) with its conjugate base (A ). - If an acid such as HCl is then added to such a solution, A can neutralize it, in the process being converted to HA. - If a base is added, HA can neutralize it, in the process being converted to A. a buffer as a solution that consists of a mixture of a weak acid and it conjugate base Amino acids have both a carboxylic acid group (acid) and an amine group (base). Those two functional groups can act as "pseudo" buffer sites. If an acid is added, the amine can pick up the extra protons, and if a base is added, the carboxylic acid group can sort of neutralize it. But amino acids, by themselves, do not fit the definition of a buffer. if a base is added d If an acid is added ١٦

17 Q. Draw the fully protonated structure of Alanine? 17

18 Learning Check AA2 + CH 3 CH 3 H 3 N CH COOH H 2 N CH 2 COO (1) (2) Select from the above structures A. Alanine in base. B. Alanine in acid. 18

19 Solution AA2 + CH 3 CH 3 H 3 N CH COOH H 2 N CH 2 COO (1) (2) Select from the above structures A. (2) Alanine in base. B. (1) Alanine in acid. 19

20 Learning Check Would the following ions of serine exist at a ph above, below, or at pi? O O O H 3 N CH C O H 3 N CH C OH H 2 N CH C O CH 2 CH 2 CH 2 OH OH OH The anser. PH=PI PH < PI PH >PI 20

21 6.Spectroscopic Properties All amino acids absorb in the infrared (bond vibrations). Only Phe, Tyr, and Trp absorb in the UV (electronic transitions between energy levels). Absorbance at 280 nm is a good method for determining protein concentration.. The UV spectra of the aromatic amino acids at ph 6. Beer s Law: A = εcll 21 21

22 Absorption of light by molecules Spectrophotometer Wave length of light. Ultrviolet nm Visible Infra red

23 Chemical reactions Due to carboxyl group: 1.amino acids form salts (COONa) with bases & ester (-COOR ) with alcohol. 2. Decarboxylation: form corresponding amine. 1. Histidine histamine + CO 2 2. Tyrosine tyramine + CO 2 3. Tryptophan tryptamine + CO 2 4. Lysine cadaverine + CO 2 5. Glutamic acid GABA + CO

24 24

25 AMINO ACIDS The building blocks of proteins Released from proteins by hydrolysis (e.g. with 6M HCl, 110ºC, 24 h) Separated by chromatography or electrophoresis Detected by reaction with ninhydrin (purple colour) 25

26 Separation and Analysis using pi values Differences in isoelectric points (and therefore charges) are used to separate mixtures of amino acids by two common methods: - Ion exchange chromatography :Ion exchange resins have charged groups covalently attached to the stationary phase (adsorbent, matrix), either positive or negative. Obviously, if ionizable groups are weak acids or bases, the ph of the buffer determines the charge state of the matrix. - Electrophoresis Mixture of: buffered at ph 6.0 H 3 H O H O H O N C CO + H 3 N C CO + H 3 N C CO CH 2 CO 2 CH 3 CH 2 CH 2 CH 2 CH 2 NH 3 D (pi=2.8) A (pi=6.0) K (pi= 9.7) aspartic acid alanine lysine 26

27 Ion Exchange Chromatography Mixture of: buffered at ph 6.0 H 3 N H O H O H O C CO CH 2 CO 2 D (pi=2.8) + H 3 N C CO CH 3 A (pi=6.0) + H 3 N C CO CH 2 CH 2 CH 2 CH 2 NH 3 K (pi= 9.7) SO 3 K (strongly retained) sulfonated polystyrene SO 3 A (slightly retained, D (unretained) & SO 3 D - elutes first, followed by A; K + elutes last, and on lafter ph of buffer is increased and K + is deprotonated. 27

28 But there is a problem in detecting amino acids; they are colorless, l and most of them have very little absorption in the UV region (they have no conjugation, except in the four aromatic amino acids) To overcome this difficulty, amino acids are converted (after separation by ion exchange chromatography) to a derivative using ninhydrin. 28

29 Separations of Amino Acids

30 - Q. Mixture of Val (pi = 6) ; asp ( pi = 2.8,) lys (pi = 9.7) in a buffer at ph = 6. What is the form of each? O + H 3 N C _ O CH CH O C H 3 C CH val CH 3 - O C N H 3 + CH 2 asp O - O N H 3 + H 2 C CH 2 lys N H 3 + CH C O H 2 C CH 2 O - Consider THREE amino acids mixed together in a buffer at ph 6.0 Ninhydrin is used to detect the individual amino acids 30

31 Separation of Amino Acids by Paper Chromatography h A mixture of amino acids can also be separated on the basis of polarity

32 Start Chromatography Solvent Front x Origin -Identify unknown amino acids using biochemical tests and R f values 32

33 Chromatography h Solvent Front x x Origin 33

34 Question 1.At ph 7, which of the following amino acids have a net positive charge, which have a net negative charge, and which are neutral? Lysine Phenylalanine Leucine 2- Explain How: a molecule's charge (amino acids) changes with the ph of its surroundings. 34

35 35