BIOCHEMISTRY - Laboratory Practical - NTU 2015

MODULE IV

METABOLISM OF MAJOR CLASSES OF BIOMOLECULES

Laboratory Work 10

EXPERIMENTAL VERIFICATION OF AMINO ACID METABOLIC CONVERSIONS

Objective: to verify the enzymatic conversions of Amino Acids using model experiments.

Basic Theoretical Background

METABOLISM is a set of biochemical reactions that sustain living organisms. Metabolism is divided into anabolism and Catabolism. During anabolism, Peptides and Proteins are synthesized from amino acids. The common catabolic reactions for amino acids include decarboxylation, deamination, Transamination, and transpeptidation. As a result of decarboxylation, an amino acid loses its carboxyl group in the body, CO₂ is released, and a biogenic amine is formed.

Deamination is classified into four types — intramolecular, reductive, hydrolytic, and oxidative — which yield ammonia and an unsaturated carboxylic acid, a saturated carboxylic acid, an α-hydroxy acid, or an α-keto acid, respectively. Oxidative Deamination is the most prevalent in living organisms.

Transamination (indirect deamination or transpeptidation) is a reaction between an α-Amino Acid and an α-keto acid, resulting in The formation of a new α-amino acid and an α-keto acid without the intermediate release of ammonia.

The reaction is catalyzed by aminotransferases (transaminases), whose Coenzymes are Pyridoxal phosphate (PLP) and pyridoxamine phosphate (PMP). Aminotransferases are found in most animal and plant Tissues, as well as in microorganisms.

Equipment: test tube rack, Glass stirring rods, capillaries, pipettes, glass funnels, flasks, Water bath, dropper bottles, thermostat, gas burner, Chromatography paper, paper filters, Silufol chromatography plates, Petri dish for radial chromatography or a glass beaker for ascending chromatography, spray bottle, stopwatch, laboratory centrifuge, photoelectric colorimeter (PEC).

10.1. Transamination between Alanine and α-ketoglutaric acid

Materials and Reagents: enzyme preparation (5 g of brewer's Yeast ground in a porcelain mortar with 2–3 ml of distilled water and a small amount of quartz sand, mixed with 8–10 ml of water, and filtered through cotton wool), 1% alanine solution, 1% glutamic acid solution, 1% pyruvic acid or sodium Pyruvate solution, 1% α-ketoglutaric acid solution, 0.1% potassium carbonate solution, 0.025% monobromoacetic acid solution, 10% trichloroacetic acid solution, freshly prepared ninhydrin reagent (9.5 ml of 0.5% ninhydrin solution in 95% acetone, 0.1 ml of glacial acetic acid, and 0.4 ml of distilled water), n-butanol–glacial acetic acid–water mixture (4:1:5), concentrated KOH solution (100 g of KOH dissolved in 60 ml of distilled water), 2% alcoholic solution of salicylaldehyde.

Procedure

The transamination process between alanine and α-ketoglutaric acid is catalyzed by the enzyme alanine aminotransferase (ALT), producing pyruvic acid (PA) and glutamic acid. The reaction proceeds with the participation of PLP and PMP; no ammonia is released, and substituted Imines or Schiff bases are formed as reaction intermediates. During the reaction, the levels of alanine and α-ketoglutarate decrease, while glutamic acid and pyruvate are formed:

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Add 0.5 ml of alanine solution, 0.5 ml of α-ketoglutaric acid solution, 1 ml of K₂CO₃ solution (to achieve optimal pH), and 0.25 ml of monobromoacetic acid solution (to prevent Glycolysis and inhibit the reduction of pyruvate to lactate) into two test tubes. Add 1 ml of the active enzyme preparation to the test tube, and 1 ml of the enzyme preparation boiled for 2–3 minutes to inactivate the Enzymes (followed by cooling) to the control tube. Stopper both the test and control tubes and incubate them in a thermostat at 37 °C for 1 hour. To precipitate proteins, add 0.25 ml of 10% trichloroacetic acid solution to each tube and filter the mixtures.

The presence of pyruvate is confirmed by a reaction with salicylaldehyde. The contents of alanine and glutamic acid in the filtrate are determined chromatographically.

Reaction for Pyruvic Acid

To 2 ml of the filtrate from each tube, add 2 ml of KOH solution and 1 ml of salicylaldehyde solution, shake the tubes, and incubate for 10–12 minutes at 37 °C. The test tube develops an intense orange-yellow color, indicating the formation of PA. In the control tube, the color remains light yellow.

To construct a calibration curve, add 0.2, 0.4, 0.6, 0.8, and 1.0 ml of the PA solution into 5 test tubes, and bring the volume up to 2 ml with water. Add 2 ml of KOH solution and 1 ml of salicylaldehyde solution to each tube, shake, and incubate for 10–12 minutes at 37 °C.

Measure the optical density of the solutions against distilled water using a PEC with a blue light filter at a wavelength of
450–480 nm.

Radial Paper Chromatography

Chromatographic paper is cut into a circle with a diameter 0.5 cm larger than that of a Petri dish. A strip is cut toward the center of the circle and bent downward. The circle is divided into 4 segments using a pencil. At a distance of 0.5 cm from the center, a start line is marked, and one of the solutions (test sample filtrate, control sample filtrate, standard alanine or glutamic acid solution) is applied dropwise with a capillary 2–3 times within the segments, carefully drying the paper over a burner each time. A mixture of n-butanol, glacial acetic acid, and water is poured into the bottom of the Petri dish, the paper is immersed into the liquid with its cut strip, and the dish is covered. Chromatography is stopped when the solvent front reaches almost the edge of the paper (0.5–1 cm from the edge). The paper is removed, and the solvent front is immediately marked with a pencil.

The chromatogram is developed as follows: it is carefully dried over a burner, treated with a ninhydrin solution, and dried in a drying oven at 70 °C for 10–15 min. The colored spots are marked with a pencil. The R_f value for each spot is calculated as The ratio of the distance traveled by The amino acid to the distance traveled by the solvent, and the positions of the spots obtained from the control and test filtrates are compared with those of alanine and glutamic acid. The sizes of the spots are also compared.

Thin-Layer Chromatography

A 4×10 cm rectangle is cut from a Silufol plate. At a distance of 0.5 cm from the lower edge of the plate, a start line is carefully drawn with a pencil, and standard amino acid solutions as well as test solutions are applied to it at separate points using a capillary 2–3 times, drying after each application. The plate is immersed in a beaker containing a mixture of n-butanol, glacial acetic acid, and water so that the start line is not submerged in the liquid. The beaker is covered with a glass lid, and the solvent front is allowed to rise until it is 0.5 cm away from the upper edge of the plate. The plate is then removed, and the solvent front line is marked with a pencil.

The chromatogram is developed and evaluated in the same manner as for radial paper chromatography.

To determine the absolute decrease in alanine content, known quantities of alanine and the test filtrates are applied to the plate or chromatographic paper. Using one of the quantitative amino acid Determination Methods (visual comparison, densitometry, or elution from chromatograms), the alanine content in the test and control filtrates is determined. By analyzing the obtained results, the decrease in alanine content in the sample compared to the control is established.

10.2. Determination of Aspartate Aminotransferase
and Alanine Aminotransferase Activity in Blood Serum by Reitman
and Frankel

Materials and Reagents: blood, 0.1%
2,4-dinitrophenylhydrazine (DNPH) solution, 0.4 M NaOH solution, 1 µM pyruvate solution, substrate-buffer mixtures. (To determine AST activity, 29.2 g of α-ketoglutaric acid and 2.66 g of D,L-aspartic acid (1.33 g of L-aspartic acid) are dissolved in 1 M sodium hydroxide solution until the precipitate is completely dissolved (pH 7.4); the solution is transferred into a 100 ml volumetric flask, brought to the mark with 0.1 M phosphate buffer (pH 7.4), supplemented with 1 drop of chloroform, and stored frozen in a refrigerator. To determine ALT activity, the substrate-buffer mixture is prepared similarly, substituting 1.78 g of D,L-alanine (0.89 g of L-alanine) for aspartic acid).

Procedure

The method is based on the determination of pyruvic acid (pyruvate, PA), which is either one of the products of the reaction catalyzed by ALT or the product of decarboxylation of oxaloacetate (oxaloacetic acid, OAA) formed in the reaction catalyzed by AST:

α-Ketoglutarate + Alanine ↔ Glutamate + Pyruvate

α-Ketoglutarate + Aspartate ↔ Glutamate + Oxaloacetate

Oxaloacetate → Pyruvate + CO₂

The resulting pyruvic acid is determined via a color reaction with 2,4-dinitrophenylhydrazine:

Blood is incubated for 30 min at 30 °C in a water bath, and then centrifuged for 15 min at 3,000 rpm. The supernatant is used to determine enzyme activity.

Determination of AST Activity

0.5 ml of the substrate-buffer mixture containing aspartate is added to a test tube, incubated in a thermostat for 5 min at
37 °C, and 0.1 ml of blood serum is added, followed by incubation for 60 min at 37 °C. Then, 0.5 ml of DNPH solution is added and left for 20 min at room Temperature (18–25 °C). Next, 5 ml of sodium hydroxide solution is added, mixed thoroughly, and incubated for 10 min at room temperature. The optical density is measured on a photocolorimeter using a green light filter (wavelength 500–560 nm) in a 1 cm cuvette.

The control sample is prepared in the same way as the test sample, except that the blood serum is added after the 60 min incubation at 37 °C and the subsequent addition of DNPH.

Determination of ALT Activity

0.5 ml of the substrate-buffer mixture containing alanine is added to a test tube, incubated in a thermostat for 5 min at 37 °C, 0.1 ml of blood serum is added, and the mixture is incubated for 30 min at
37 °C. Subsequent steps are carried out in the same manner as for AST.

Construction of the Calibration Curve

To construct the calibration curve, 0.1, 0.2, 0.3, and 0.5 ml of the pyruvate solution are taken, brought up to 0.6 ml with water, and 0.5 ml of the DNPH solution is added. The samples are then processed identically to the control and test samples. A graph of optical density versus pyruvate content in the sample (µmol) is plotted.

Calculation of Enzyme Activity

AST and ALT activities are expressed in micromoles of pyruvate formed during the incubation of 1 ml of blood serum at 37 °C for
60 min. The amount of pyruvate is determined from the calibration curve, multiplied by 10 to convert to 1 ml of serum, and for ALT, further multiplied by 2 to account for the 60-min incubation period.

Physiological values of aminotransferase activity per 1 ml of blood serum after incubation at 37 °C for 1 h: AsAT — 0.1–0.5 µmol/(h·ml); AlAT —
0.1–0.7 µmol/(h·ml).

Processing of experimental data

Construct calibration curves to determine the pyruvic acid content in the solution and to determine the aminotransferase activity in blood serum. Calculate the PA content and The activity of AsAT and AlAT. Compare the results of radial paper chromatography and thin-layer chromatography.

Selection/5.html">Control Questions and tasks

1. Write the transamination reaction between aspartic acid and pyruvate or α-ketoglutarate. Name the enzyme, coenzymes, and reaction intermediates. Explain the relationship between transamination and deamination in the body.

2. Write the decarboxylation reactions of Histidine, Lysine, and the α-decarboxylation of glutamic acid. Name the enzymes, coenzymes, and reaction products, and explain their biological role.

3. Write the deamination reactions of alanine (4 types). Name the enzymes, coenzymes, and reaction products.

4. Name the Pathways of ammonia formation and detoxification in the body. What are the End products of Protein metabolism?

5. In which anabolic reactions do amino acids participate?

6. Write the reaction of Glutathione formation and explain its biological significance.

References: [1; 3—7].