1. IDENTIFICATION AND DETERMINATION OF ISOELECTRIC POINT OF AMINO ACID

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1 1. IDENTIFICATION AND DETERMINATION OF ISOELECTRIC POINT OF AMINO ACID The practical training follows the lectures: Metabolism of amino acids, Biosynthesis and degradation of proteins and 1 st year seminar dedicated to the analytical methods and ph. For a successful completion of this practical training it is necessary to have 1 page of millimetre paper and 2 rulers one setsquare at least for each group (2 students)! The necessary part for your procedure in this practical training is page Clinical and pathobiochemical aspects of metabolism of amino acids Functions of amino acid in organism are very different protein structure, carbon skeleton can serve as a source of energy, conversion to other important products such as hormones, signalling molecules etc. The organism is unable to store amino acids in period of energy deficit, contrary to molecules of lipids and carbohydrates. The role of amino acids and proteins is not primarily nutritional. Catabolism of amino acids is very energy consuming metabolic pathway running exclusively in the liver (ureosynthetic cycle). There is a number of pathological conditions associated with metabolism of amino acid. It concerns different enzymopathy with lack of enzyme needed for synthesis or degradation of amino acid. High accumulation of metabolites can impair, for example, brain in phenylketonuria. Patients (children) with poorly developed blood-brain barrier are exposed to toxic metabolites and threaten serious damage of development and function of neural tissue. In other cases, there is a lack of transporter for absorption or excretion of amino acids. There is a plenty of disorders of ureosynthetic cycle. Absence or deficit of enzymes leads to inability to eliminate amine nitrogen from organism and increased ammonia levels in blood with the development of potentially fatal toxic encephalopathy. The knowledge of amino acid structure and metabolism is necessary to explain a number of pathophysiological relations and for understanding of syndromology of this group of diseases. 2. ELECTROCHEMICAL CHARACTERISTICS OF AMINO ACIDS Amino acids are organic acids containing at least two dissociable functional groups one acidic carboxylic (-COOH) and one basic amino group (-NH2). Biogenic amino acids bind this two functional groups exclusively on α-carbone. Some of biogenic amino acids can bind another dissociable groups on subordinate chain (-R). Acid-base equilibrium in water solutions depends on ph: R NH 3 + pk a1 R NH 3 + pk a2 R NH 2 HO O O - O O - O In an acidic conditions (exactly ph < pi) both groups bind hydrogen cation. Rising ph releases the carboxylic hydrogen cation firstly, because of the lower value of dissociation constant. Hydrogen cation in amino group does not dissociate and a so-called zwitterion is generated, which carries both positive and negative charge. Further increasing of ph (ph > pi) causes separation of cation from amino group and both functional groups exist as conjugate bases. 1/9

2 2.1 Isoelectric point of amino acids Isoelectric point of amino acids is value of ph, when amino acid has neutral electric charge (zwitterion) and doesn t move in direct-current electric field. Isoelectric point of amino acids, which contain two dissociable functional groups, can be calculated with satisfactory accuracy as an arithmetic mean of dissociation constant: pk pk a1 a 2 pi 2 3. THIN LAYER CHROMATOGRAPHY IN AMINO ACIDS SEPARATION Thin Layer Chromatography (TLC) is an analytical method using different distribution of substances between mobile (solvent) and stationary (sorbent on inert plate) phase. Owing to different distribution of substances between mobile and stationary phase, we obtain different migration of the substances along the desk. Thus the substances are separated according to their polarity. We next use, for example, ninhydrine for highlighting resolved amino acids. The colours range from blue to brown, prolin and hydroxyproline are yellow. 4. POTENTIOMETRIC TITRATION OF AMINO ACID Direct potentiometry is an electrochemical method, in which we measure electromotive voltage of a pile. The pile consists of a specific (indicative) and a referent electrode. The potential of a specific electrode depends on activity or concentration of determined ion. The potential of a referent electrode is independent of the composition of the solution it has a constant value. Potentiometry is a frequently used method for objective determination of an end-point of titrations. In potentiometric titrations, the values of electromotive voltage or ph are registered after each addition of titration solution. The strongest response of specific electrode is around the point of equivalence. We can use either graphic or numeric method for data evaluation. 1. Graphic methods: Method of parallel tangent Linear method (method of bisector) 2. Numeric method: Method of first and second derivation 2/9

3 5. IDENTIFICATION OF UNKNOWN AMINO ACID BY TLC On your desk you will find a solution containing an unknown amino acid. Identification should be carried out by comparing the sample retention factor with the retention factors of standards. Procedure: 1. Mark start on the shorter side of chromatographic plate 2 cm from the bottom and finish 1 cm from upside. And right and left border 0,5 cm from the lateral end of desk. Use a soft pencil. 2. Put the standards of amino acids and unknown sample on start via dropper. The diameter of splotch must be smaller than 5 mm. Let the drops become dry. 3. Put the chromatographic plate into the beaker with distributing solution (ethanol: water, 7: 3) so that the mobile phase almost reaches the start and let the solvent move up the plate by capillary action. 4. Stop the chromatography when the mobile phase reaches your marked finish. 5. Dry the chromatographic plate and then put the desk into the solution of ninhydrin for a moment. Finally put the desk into the drier (90 C) for 5 min. 6. On the dried plate mark the centre of a splotch and measure the distance between start and centre of a splotch (a), distance between start and finish (b) and calculate retention factors Rf (Rf = a/b). 7. Compare values of retention factors of unknown samples and standards and define the unknown amino acid. 6. POTENTIOMETRIC TITRATION OF UNKNOWN AMINO ACID To monitor the changes in ph depending on added volume of titration solution we use ph-meter. Before each analysis it is necessary to calibrate the ph-meter. Solid sample of unknown amino acid is on your desk. The sample contains the same amino acid as the solution tested by TLC. Procedure: 1. In to the beaker (100 ml) weigh g of unknown amino acid and this amount dissolve in approximately 35 ml of distilled water. Mix the solution using magnetic stirrer so long as whole amino acid is dissolved. 2. Measure the ph using standards (ph = 4 and ph = 7) and calibrate the ph-meter if stable ph on display differs of about 0.1 from standard. 3. Acidify the solution of amino acid by 1 M HCl so that final ph is about 2. Add the acid by 0.5 ml (maximal added volume is 2.5 ml). 4. Titrate acidic solution of amino acid by solution of 0.3 M sodium hydroxide. Add 0.5 ml of the hydroxide by burette and detect the ph (wait for stable value). Titrate till ph is changing very slightly (above ph ~ 12,3). 5. After each addition of hydroxide wait for ph stabilization. It can take a few minutes for ph to stabilize in a zone of point of equivalence. 6. Record your values on millimetre paper volume of added hydroxide on horizontal axis, values of ph on vertical axis. Link the points together and analyse the titration curve by method of 3/9

4 parallel tangents or by linear method and determine the pi value of unknown amino acid. Compare your results with tabulated values. Amino acid pka1 pka2 pka3 pi Phe ,91 Hy-Pro ,74 Gly Met Leu RESULTS 7.1 TLC Amino acid a b Rf color Phe Gly Met Hy-Pro sample Identified amino acid: 7.2 POTENTIOMETRIC TITRATION OF UNKNOWN AMINO ACID V[ml] ph V[ml] ph V[ml] ph Calculated values: pka1 = pka2= 4/9

5 pi = pitab= Discussion: 1. Try to explain the development of toxic encephalopathy in patients with urea cycle disturbances (use the textbooks, internet...). 2. How can the composition of amino acids influence the final structure and function of the protein? 3. Draw the protonation equilibria of histidine in water solution via the formulas and calculate ph for each form. Histidine concentration in water solution is c(his) = 0.01 mol/l. H 2 His 2+ pk a1 =1,70 pk a2 =6,02 pk HHis + a3 =9,08 His His - 4. Normal hemoglobin has pi= Hemoglobin S in sickle cells has pi= Try to explain the effect of this change on hemoglobin physiological functions. Conclusion: 5/9

6 APPENDIX 1: ANALYSIS OF TITR. CURVE BY PARALLEL TANGENT METHOD Good identification of a point of equivalence is the basis for data evaluation. Titration of amino acids is titration of a weak acid by a strong basis. For identification of a first point of equivalence we could use the visual acid base indicator. But for the second point of equivalence it is necessary to use a potentiometric detection. We need to know whole the titration curve. For that reason we measure the changes of ph after each hydroxide addition. The curve has typical S shape with a point of equivalence in inflection point. Procedure: 1. Draw the tangent t1 in the beginning abrupt part of curve. 2. Draw the tangent t2 parallel with t1 on the second part of titration curve (fig. 1). 3. Mark the half-length between t1 and t2 and draw the third tangent. Fig. 1 Tangent construction. 4. Read the values of volume and ph in intersection of curve and tangent t3. These are the values of ph and volume at the the point of equivalence (Fig. 2). 6/9

7 Fig. 2 Subtraction of ph and volume values. 5. Determination of volume in second point of equivalence is analogous. V1 = 3,8 ml and V2 = 7,4 ml are results from our example. 6. The values of pka1 and pka2 are calculated from Henderson Hasselbalch s equation as the ph value in half phase of titration (Fig. 3). It means that ph value is in accordance with V/2. Fig. 3 Detection of pka values. 7/9

8 APPENDIX 2: TITRATION CURVE ANALYSIS BY BISECTOR METHOD Bisector method differs from parallel tangent method only in equivalence point identification. Fig. 4 Bisector construction. Smooth the titration curve with suitable bisector. Restricted areas in both parts of curve must be the same. Further data processing is analogous to a described procedure in supplement 1. 8/9

9 1. Hydrochloric acid APPENDIX 3: MATERIAL SAFETY DATA SHEET Risk phrases: H314 Causes burns. H335 Irritating to respiratory system. Safety phrases: H314 Causes burns. P280 Wear suitable protective clothing, gloves and eye/face protection. 2. Sodium hydroxide Risk phrases: H314 Causes burns. Safety phrases: P280 Wear suitable protective clothing, gloves and eye/face protection. 9/9