FUNDAMENTALS OF ENZYME STRUCTURE AND KINETICS IN BIOLOGICAL SYSTEMS - O. A. Naumenko - 2017

4 Laboratory Workshop

4.1 Performing protein precipitation reactions and evaluating their Primary and secondary composition using color reactions

Protein precipitation reactions are employed in the Separation of protein fractions during protein Isolation and Purification, for obtaining protein-free solutions (e.g., when isolating DNA from animal Tissues), and for the rapid detection of Proteins in biological fluids (such as urine).

Altering the charges of protein molecules and/or stripping their Hydration shell leads to the aggregation of protein molecules and their precipitation, known as Protein Denaturation. Upon heating, protein molecules denature—meaning they lose their native Structure and unfold—while remaining in suspension, which is visually observed as the turbidity of a diluted protein solution.

Objective

To carry out precipitation reactions of protein solutions using various Methods.

To evaluate the qualitative composition of protein solutions using color reactions both before and after the precipitation Procedures.

Principle of the method

To precipitate a protein, it is necessary to eliminate its stabilizing factors: neutralize its charge, remove its hydration shell, or disrupt its tertiary structure (induce denaturation) by applying various chemical Reagents or heating.

Guidelines for Laboratory Work

1. Heating induces protein denaturation, accompanied by solution turbidity.

2. Mineral and organic acids cause dehydration and a shift in the charge of some protein molecules, accompanied by their denaturation.

3. Heavy metal salts cause protein denaturation due to the adsorption of Metal Ions onto The surface of the protein molecule and The formation of an insoluble complex.

4. Organic Solvents strip the hydration shell and reduce protein stability in solution, though precipitation occurs only in neutral and slightly acidic solutions. Prolonged exposure to an organic solvent leads to protein denaturation.

5. The color intensity in color reactions is proportional to The amount of reacting functional groups. Therefore, color reactions can be used for the qualitative and Quantitative determination of proteins, for identifying constituent Amino acids, or for analyzing protein composition:

- the biuret test is used to detect peptide bonds in oligopeptides and proteins (a universal test for all proteins);

- the ninhydrin test is used to detect α-amino groups in free amino acids, oligopeptides, and proteins (a universal test for all proteins);

- the xanthoproteic test is used to detect aromatic amino acids in proteins;

- the Fohl's test is used to detect the sulfur-containing amino acid Cysteine in proteins.

Procedure

1. Preparation of protein solutions.

Solution I: the white of one chicken egg is diluted in 300 ml of distilled Water and filtered; Solution II is a 1% gelatin solution. Solution I contains Ovalbumin (egg albumin), while Solution II contains gelatin (ovalbumin contains all 20 amino acids, whereas gelatin contains only 17).

2. Perform color reactions with each protein solution and record the results in Table 4.1.

Class="center">Table 4.1 — Color reactions

3. Perform protein precipitation using various methods and record the results in the table (Table 4.2):

Table 4.2 — Protein precipitation reactions

4. Perform color reactions with protein solutions after precipitation.

5. Formulate your Conclusions.

Review Questions for Laboratory Work

1. Where are protein precipitation reactions applied in practice?

2. What happens to the Cell/13.html">Protein Structure during its precipitation?

3. Describe The Mechanism of the biuret reaction.

4. Describe the mechanism of the xanthoproteic reaction.

5. Describe the mechanism of Folin's reaction.

6. Describe the mechanism of the ninhydrin reaction.

7. Will egg albumin give positive biuret and ninhydrin reactions after precipitation with heavy metals? Explain why.

8. Will Folin's reaction be positive for egg albumin after precipitation with heavy metals, and why?

4.2 Quantitative Determination of protein by the Biuret Method

Protein concentration determination is carried out during the study, isolation, and purification of proteins; in industry during The production of Enzymes, dietary proteins, and protein-based Pharmaceuticals.

There are many different Methods for the quantitative determination of proteins. Among them, colorimetric Methods based on the ability of proteins to form colored complexes with A number of reagents (color reactions) have become widespread. The most common among these methods are the biuret method and its modification (the Lowry Method).

Objective

Master the operation of a photoelectric colorimeter (PEC). Learn how to construct standard calibration curves. Determine the protein content in a sample of unknown concentration.

Principle of the Method

The biuret method is based on the formation of a violet-colored coordination complex between nitrogen atoms involved in the peptide bonds of the protein and divalent copper ions in an alkaline medium (biuret reaction). The intensity of the developing color of the solution is directly proportional to the protein concentration and is determined photometrically. For Quantitative Protein Determination, the color intensity of standard protein solutions with a known concentration is measured (optical density value $D$). Based on the obtained data, a graph of the optical density of the colored solutions versus their protein concentration is plotted (standard calibration curve). The optical density value is always plotted on the ordinate axis as a function of the set values.

Measure the optical density of the colored solution with an unknown protein concentration and use the standard curve to determine the protein concentration in it. A serum albumin solution is typically used as a standard protein solution.

Procedure

1. Prepare the biuret reagent from the stock concentrate (copper sulfate – 120 mmol/L, potassium iodide – 300 mmol/L, potassium sodium tartrate – 320 mmol/L, sodium hydroxide – 3 mol/L) and dilute it immediately before use in a 1:19 ratio.

2. Prepare solutions of three concentrations:

- 0 mg/mL;

- 20 mg/mL;

- 30 mg/mL.

3. Add the solutions to three test tubes as outlined in Table 4.3, mix thoroughly, and incubate for 30 minutes at room Temperature.

Table 4.3 - Steps for quantitative protein determination

4. Perform photoelectric colorimetry against H₂O at a wavelength = 545 nm and a cuvette path length of 1 cm, and determine the optical density value D_t for each solution.

5. Construct a calibration curve showing the optical density of the colored solutions as a function of their protein concentration. The ordinate axis is always used to plot the value O as a function of the specified values.

6. Measure the optical density of the solution with an unknown protein concentration and determine its protein concentration using the standard curve.

Review Questions for the Laboratory Work

1. Where is quantitative protein determination applied in practice?

2. How to prepare the biuret reagent.

3. What color reactions form The basis of quantitative protein determination, and why?

4. How to construct a calibration curve?

4.3 Dialysis of a Protein Salt Solution

Dialysis is The process of separating high-molecular-weight substances (such as proteins) from low-molecular-weight substances (such as salts) using semipermeable membranes.

Semipermeable membranes are those whose pore diameter allows only low-molecular-weight compounds to pass through. Examples of natural semipermeable membranes include Bowman's capsules in the Kidneys. Artificial semipermeable membranes such as cellophane and collodion are widely used and serve as the basis for dialyzers, including the "artificial Kidney".

The dialysis method is utilized in scientific research laboratories, industry, and clinical practice.

Objective

To prove that the low-molecular-weight compound CaCl₂ passes through a semipermeable membrane, whereas the high-molecular-weight protein does not and remains within the dialyzed solution.

Principle of the Method

The method is based on the fact that low-molecular-weight substances readily diffuse through semipermeable membranes into a pure solvent, forming a dialysate. Diffusion will continue until the concentrations of the diffusing substance are equalized between the dialyzed solution and the dialysate. The process will resume if the dialysate is replaced with a fresh solvent or if this replacement is carried out continuously (continuous-flow dialysis).

Procedure

1. Preparation of the dialyzer. Dialysis tubing or cellophane dialysis bags can be used as semipermeable membranes. A piece of cellophane (a 10x10 cm square) is moistened with distilled water, shaped into a pocket, and thus formed into a dialysis bag.

2. Preparation of the dialyzable solution. Place 20 drops of egg white diluted in a 5% KCI solution (the white of one egg is diluted in 300 ml of a 5% KCI solution) into a dialysis bag and mix. Clamp the edges of the cellophane between two Glass rods secured together with rubber rings.

3. Dialysis. Place the bag containing the protein saline solution into a beaker filled with distilled water so that the portion of the bag containing the protein solution is fully submerged in the water. Immediately, streams of the saline solution flowing down from the bag to the bottom of the beaker can be observed against the light, which is caused by A change in water refraction.

4. Analysis of dialysis results. 60 minutes after starting the dialysis, perform qualitative tests for protein (biuret test) and chloride ions (with AgNO₃) in accordance with Table 4.4.

Table 4.4 - Procedure for conducting the study

5. Formulate your conclusions.

Control Questions for the laboratory work

1. Where does protein dialysis take place in The Human Body?

2. What can serve as a dialyzer?

3. What are semipermeable membranes?

4. How are dialyzable solutions prepared?

5. Which reactions confirm the efficiency of dialysis and why?

6. What is continuous-flow dialysis?

4.5 Paper Chromatography of amino acids

The chromatographic method, developed by the Russian scientist M. S. Tswett, is one of the relatively simple and rapid methods for separating mixtures of substances.

There are several variations of this method depending on THE PRINCIPLE OF separating a mixture into its constituent components (adsorption, ion-exchange, and Affinity Chromatography).

An example of adsorption chromatography is paper partition chromatography, which has proven to be the most convenient for separating amino acids that differ in the Hydrophobicity of their side chains, as well as for

identifying unknown amino acids by comparing their mobility with the mobility of known reference amino acids ("standards").

Objective of the work

Perform the separation of a mixture of two unknown amino acids, calculate the partition coefficient (R_f) for each of them, and identify the amino acids by comparing their R_f with those of standard amino acids (glutamic acid and leucine).

Principle of the method

The method is based on the differing solubility of individual amino acids in two partially miscible liquids: water, which is held by the filter paper and acts as the stationary solvent phase, and an organic solvent serving as the Mobile phase (butanol: acetic acid: water in a 5:1:4 ratio).

As the solvent moves along the paper, multiple redistributions of the amino acids occur between the mobile and stationary solvent phases. The greater the hydrophobicity of an amino acid, the faster it will move along the paper, and vice versa. Accordingly, the distances traveled by the amino acids with the mobile solvent phase along the paper will vary.

The rate of movement of each amino acid can be expressed by its partition coefficient (R_f). The partition coefficient is defined as The ratio of the distance (in mm) from the point of application of The amino acid (the origin) to the center of its spot (value a) to the distance from the origin to the solvent front (value b). The higher the hydrophobicity of the amino acid side chain, the closer the R_f value will be to unity.

The partition coefficient for each amino acid remains constant under standard experimental conditions (paper type, temperature, solvent type, humidity) and serves as a means of identifying amino acids by comparison with reference ("standard") amino acids.

Procedure

1. Mark a 12 cm diameter chromatographic paper disc with a pencil (Figure 4.1).

Figure 4.1 - Chromatographic paper disc

2. Apply 2 µL of standard amino acid solutions (0.6% glutamic acid solution and 0.5% leucine solution) and the unknown amino acid mixture to the spotted locations. Air-dry the paper disc containing the samples.

3. Cut the chromatographic disc along the cutting lines, fold the resulting tab along the crease line, and trim it to a length of 3 cm.

4. Place the disc into the chromatographic chamber (Petri dish) so that the tab is immersed in the organic solvent.

5. Once the solvent front has traveled 5–6 cm, remove the chromatogram from the chamber and carefully outline the solvent front with a pencil.

6. Dry the disc in a drying oven at a temperature of 60 to 80 °C.

7. Immerse the disc in a 0.5% ninhydrin solution in acetone and dry again until the amino acid spots appear.

8. Calculate the $R_f$ value for each amino acid, and then paste or sketch the resulting chromatogram into your notes.

9. Draw your own conclusions independently.

Review Questions for Laboratory Work

1. What is chromatography?

2. What are the Different types of chromatography?

3. What category of chromatography does paper partition chromatography belong to?

4. What is the partition coefficient of an amino acid?

6. What are reference amino acids ("standards")?

7. How is a chromatographic disc prepared?

8. What is the solvent "front"?

9. What is the underlying principle of amino acid partition chromatography?

10. How is the chromatogram developed?

4.6 Titrimetric Determination of Catalase Activity

Equipment and reagents: boiling water bath; 5, 10, 20, and 25 mL pipettes; 10 and 25 mL graduated cylinders with spout; 100 mL volumetric flask; 200 mL Erlenmeyer flasks; porcelain mortar and pestle; potassium permanganate (0.1 N); sulfuric acid (10%); sodium carbonate; hydrogen peroxide (0.1 N); fresh plant material (potato or carrot).

Procedure

1) 2 g of raw potato (or carrot) is ground in a mortar, gradually adding 2-3 ml of water. To reduce the acidic reaction, sodium carbonate is added on the tip of a spatula until the evolution of carbon dioxide bubbles ceases;

2) the ground mass is quantitatively transferred into a volumetric flask and brought up to a volume of 100 ml with water;

3) the mixture is left to stand for 30 minutes and then filtered;

4) the activity is then determined According to the scheme: 2 test samples and 2 control samples, in accordance with Table 4.5:

Table 4.5 - Workflow

The test and control samples are titrated with a 0.1 N potassium permanganate solution (until a pale pink coloration stable for approximately 1 min is formed). The volume of the potassium permanganate solution used for titrating the remaining hydrogen peroxide (after enzymatic decomposition) in the test flask and for titrating the total hydrogen peroxide in the control flask is recorded. The amount of permanganate equivalent to the amount of hydrogen peroxide decomposed by the enzyme is found from the difference between the test and control titrations.

The calculation is performed in accordance with the reaction equation:

5Н₂О₂ + 2KMnО₄ + 3H₂SО₄ —> 2MnSО₄ + K₂SО₄ + 5О₂ + 8Н₂О, according to which 1 ml of 0.1 N potassium permanganate solution corresponds to 1.7 mg of hydrogen peroxide.

Calculation example: a catalase extract with a volume of 100 ml is prepared from 1.25 g of carrot; titration of the test sample requires 15.5 ml, and the control sample requires 30.2 ml of 0.1 N potassium permanganate solution. The amount of decomposed hydrogen peroxide in the sample is equivalent to (30.2 - 15.5) 14.7 ml of 0.1 N potassium permanganate solution and, consequently, equals (14.7 • 1.7) 24.99 mg. Thus, 1 g of raw carrot contains an amount of catalase capable of decomposing (24.99 • 100/ 20 • 1.25) = 99.96 mg of hydrogen peroxide, and per 1 min - (99.96:30) 3.33 mg. Since 1 µmol of hydrogen peroxide is 0.034 mg, 1 g of carrot contains (3.33: 0.034) 100 U of catalase.

Review questions for the laboratory practical

1. Calculate the catalase content in the test material.

2. Write the systematic name of this enzyme, its code according to the Enzyme Nomenclature, and describe its biological role.

4.7 Determination of amylase activity

The enzyme amylase (α-1,4-D-glucan glucanohydrolase, EC 3.2.1.1) catalyzes the Hydrolysis of Starch α-1,4-glycosidic bonds. The End products of starch hydrolysis are maltose and maltotriose. The primary sources of amylase in humans and animals are the Pancreas and Salivary Glands. Both forms of the enzyme are identical in catalytic properties and show virtually no difference in electrophoretic mobility. Amylase activity can increase in the presence of chloride ions. The pH optimum of the enzyme lies between 6.5 and 7.5. The enzyme contains one calcium ion per protein molecule. Therefore, in the presence of oxalate and citrate, an inhibition of amylase activity is observed, which is caused by the binding of Calcium Ions by these compounds.

Principle of the method

The proposed method is based on the hydrolytic Cleavage of an insoluble colored starch substrate by α-amylase, proceeding with the release of a free blue water-soluble dye. The amount of released dye per unit time is proportional to the enzyme activity.

Equipment: pH meter, spectrophotometer or photofluorimeter/photoelectrocolorimeter, centrifuge, magnetic stirrer, thermostat.

Glassware and laboratory ware: 0.1 ml pipettes - 2 pcs., 1 ml pipettes - 2 pcs., 2 ml pipettes - 2 pcs.; 50 ml volumetric flasks - 2 pcs.; 50 ml beaker; funnel.

Reagents: concentrated buffer solution (0.6 M phosphate buffer solution, pH 7, with 0.1 M NaCl); substrate (colored starch) - 0.6 g;

Preparation of working solutions. Buffer solution. 5 ml of the concentrated buffer solution is placed into a 50 ml volumetric flask and diluted to the mark with water. The pH of the buffer is checked before use.

Substrate suspension. 10 ml of the diluted buffer solution is placed into a 50 ml beaker, and 0.1 g of the substrate (colored starch) is slowly added under constant stirring on a magnetic stirrer. Stirring is continued until a homogeneous suspension is formed (15-20 min). The suspension prepared in this manner can be stored for one week at a temperature of +1 to +4°C.

Procedure

The activity of α-amylase is determined as follows. 1 ml of the substrate suspension is added to both the test and control tubes. The tubes are pre-warmed for 5 min at 37°C, after which 1.0 ml of Blood serum (or tissue supernatant in which the enzyme activity is being determined) is added to the test tube, and 1.0 ml of distilled water is added to the control tube. The Contents of the tubes are mixed and incubated for exactly 15 min at 37°C. Thereafter, 2.0 ml of precipitating solution is added to both tubes, and the mixture is stirred at room temperature for 15 min. The supernatant is separated from the resulting precipitate by centrifugation for 5-10 min at 3000 rpm, transferred into a 0.5 cm cuvette, and the absorbance (D) of the solutions from the test tube is measured against the solution from the control tube at 590 nm.

Calculation method. The activity of α-amylase (v) in the sample is determined using the following formula:

v = D x 1083 (E/L), (4.1)

where v is amylase activity and D is solution absorbance.

Review Questions for Laboratory Work

1. To which class of enzymes does amylase belong? State its Classification number according to the international nomenclature.

2. What reaction does this enzyme catalyze?

3. What serves as the substrate and what is the product of the reaction?

4. What is the underlying principle for determining the activity of this enzyme?

5. How is amylase activity calculated?

4.8 Determination of Peroxidase Activity

Principle of the Method

Peroxidase is the most widespread enzyme in plants and animals. Structurally, it belongs to heme-containing Glycoproteins. As the most common enzyme of biogenic tissues, peroxidase catalyzes single and coupled substrate oxidation reactions and serves as a component of antioxidant systems. Peroxidase belongs to the group of two-component enzymes composed of hemin—represented by protoporphyrin IX complexed with trivalent iron—and a polypeptide chain. The latter contains from 203 to 308 Amino Acids and, depending on The Nature of the isoenzyme, forms a compact tertiary structure consisting of two domains (Major and minor). The enzyme has a protein globule size of 50 A, containing about 43% α-helical regions.

Hemin is non-covalently anchored in a cleft of the polypeptide chain between the domains, held in place by Hydrophobic bonds and a salt bridge formed between the propionic acid residue of hemin and one of the amino groups of the apoprotein. Hemin can be reversibly dissociated from the protein at acidic pH values.

Oxidase substrates of the enzyme include dihydroxymaleic acid and other substances whose oxidation is accompanied by the formation of free-radical oxidation products.

In peroxidase-mediated oxidation reactions, the substrates can be inorganic and Organic compounds oxidized by hydrogen peroxide. During peroxidase oxidation, two substrate-binding sites are expressed on the enzyme surface, with a regulatory site located nearby. The peroxidase heme is oriented within the protein globule structure such that its vinyl groups face inward toward the protein moiety, while the propionic acid residues face outward and, through ionic bonds with Functional groups of the protein, participate in binding, orienting, and anchoring the heme to the enzyme apoprotein.

CARBOHYDRATES located On the surface of the enzyme protein globule perform the following Functions: they orient the enzyme on the membrane surface, protect the protein globule from the destructive action of free radicals, and increase the thermal stability of peroxidase.

In single and coupled peroxidase oxidation reactions, substrates of peroxidase can include antioxidants (ascorbic acid, dihydroquercetin, quercetin, hydroquinone, etc.). Low concentrations of antioxidants initiate peroxidase oxidation reactions, whereas high concentrations decrease the catalytic activity of peroxidase by inhibiting the enzyme. Depending on the Nature of the antioxidant, activation effects of peroxidase are observed in coupled substrate oxidation reactions, manifesting the individual METABOLISM/10.html">Mechanism of enzyme Action.

To determine peroxidase activity, inorganic and organic compounds are used that yield colored products upon oxidation by hydrogen peroxide. In this work, it is proposed to use o-dianisidine as a peroxidase substrate; the product of its peroxidase oxidation has absorption maxima at 460 and 420 nm, with molar absorption coefficients of 30 and 1 mM - 1cm - 1, respectively.

Equipment: pH meter, photoelectric colorimeter or spectrophotometer, centrifuge.

Glassware: 0.1 mL pipettes — 3 pcs., 0.2 mL pipette — 1 pc.; 5 mL pipette — 1 pc.; 50 mL flasks — 2 pcs. and 500 mL flasks — 3 pcs.; test tube — 1 pc.

Reagents: 0.1 M sodium phosphate buffer; pH 7.0; 4.3 mM

O-dianisidine; 15.4 mM hydrogen peroxide; wheat grain supernatant.

Preparation of reagents. The buffer solution is prepared from stock 0.1 M solutions of Na₂HPO₄ and NaH₂PO₄ by mixing them using a pH meter to pH 6.0.

The o-dianisidine solution (1.05 mg/mL) is prepared by dissolving 52.5 mg of the sample in 50 mL of 96% ethanol. The substance dissolves well upon heating. The solution should be colorless or slightly pink.

The potassium ferrocyanide solution (9.2 mg/mL) is prepared by dissolving 460 mg of the sample in 50 mL of distilled water.

Hydrogen peroxide — a 15.4-15.8 mM aqueous solution is prepared using a spectrophotometer with an absorbance of 1.12-1.15 conventional units at a wavelength of 230 nm (e = 72.7 M 1cm - 1).

Control Questions for the Laboratory Work

1. To which class of enzymes does peroxidase belong? State its EC (Enzyme Commission) number according to the international nomenclature.

2. What is the Structural Organization of this enzyme?

3. What are the substrates for this enzyme?

4. What is the principle underlying the determination of peroxidase activity?

5. How is peroxidase activity calculated?

4.9 Effect of Medium pH on Peroxidase Activity

Principle of the Method

Various environmental factors (such as pH and temperature), as well as the presence of activators and inhibitors, can significantly influence enzyme activity.

According to NMR spectroscopy data, the fifth axial iron Ligand is the imidazole group of the Histidine residue (His42) in the apoprotein. The protonation and deprotonation of the histidine imidazole (pKa ~ 6.0), which is part of the enzyme's Active Site, affect its catalytic properties at different pH levels. The optimal enzyme activity occurs in the acidic pH range and depends on the nature of the substrate.

Equipment: pH meter, photoelectrocolorimeter, centrifuge.

Glassware: 0.1 mL pipettes — 2 pcs., 0.2 mL pipette — 1 pc., 5 mL pipette — 1 pc., 10 mL pipette — 1 pc.; 100 mL flasks — 6 pcs. and 500 mL flasks — 5 pcs.; test tubes — 6 pcs.

Reagents: 0.1 M sodium acetate buffer (pH 4.0, 5.0, 6.0) and 0.1 M sodium phosphate buffer (pH 6.0, 7.0, 8.0); 1 M aqueous HCI solution; 4.3 mM hydroalcoholic solution of o-dianisidine; 15.4 mM hydrogen peroxide solution, wheat grain supernatant.

Preparation of Reagents. Sodium acetate Buffer solutions (pH 4.0, 5.0, 6.0) are prepared from stock 0.1 M aqueous solutions of CH₃COOH and CH₃COONa, while sodium phosphate buffer solutions (pH 6.0, 7.0, 8.0) are prepared from stock 0.1 M aqueous solutions of Na₂HPO₄ and NaH₂PO₄ by mixing them while monitoring with a pH meter until the desired pH value is reached. A 1 M HCI solution is prepared by dissolving concentrated stock HCI in distilled water.

The o-dianisidine solution (4.3 mM ethanolic solution) is prepared by dissolving a 105 mg sample in 50 mL of 96% ethanol. The substance dissolves readily upon heating. The solution should be colorless or slightly pink.

Hydrogen peroxide (15.4–15.8 mM aqueous solution) is prepared and verified on a spectrophotometer at an absorbance of 1.12–1.15 relative units at a wavelength of 230 nm (ε = 72.7 M^-1cm^-1).

The wheat grain supernatant is obtained as described in Section 3.5.

Procedure. Sequentially add 0.2 mL of supernatant, 2.6 mL of 0.1 M buffer solutions (pH 4.0, 5.0, 6.0, 7.0, 8.0), and 0.1 mL of 4.3 mM o-dianisidine into 6 test tubes, and mix thoroughly. The reaction in all test tubes is initiated by adding 0.1 mL of 15.4 mM H₂O₂ solution. Mix again, and after 1 minute, stop the reaction by adding 1 mL of 1 M HCI solution to each tube. Then, measure the absorbance of the solutions in each tube using a photocolorimeter at 460–500 nm (green light filter). Calculate the peroxidase activity in each tube. Finally, plot a graph on millimeter paper showing the dependence of enzyme activity on pH.

Control Questions for the Laboratory Work

1. Determine the pH optimum for peroxidase activity.

2. What factors affect enzyme activity?

3. What is The Significance of the pH optimum for enzymes?

4.10 Studying the Effect of temperature and Medium pH on Trypsin Activity

Equipment and Reagents: test tubes, 10 mL pipettes, two water baths, 1 mM BApNA solutions in 0.05 M phosphate buffer at the following pH values: 9, 7.6, 6, 5, trypsin solution (10 μg/mL), 1 N HCI.

1. To study The Effect of medium pH on trypsin activity, BApNA solutions with different pH values are used, with 2 replicates and 2 controls for each parallel sample. Determine the activity according to Table 4.6:

Table 4.6 - Workflow of the Procedure

Measure the optical density at 364 nm. The specific enzyme activity is expressed in µmol of BAPNA cleaved per 1 µg of trypsin per 1 min, using a calibration curve. Plot a graph showing the dependence of trypsin activity on medium pH.

2. To study the effect of ambient temperature on trypsin activity, follow the same procedure using a BAPNA solution with pH=7.6, but incubate the test tubes:

a) at room temperature;

b) at 4 °C (incubate in a refrigerator. Use pre-cooled solutions!);

c) in a water bath at t=55 °C;

d) in a water bath at t=75 °C.

Report Contents

1. Graph of trypsin activity versus medium pH.

2. Graph of trypsin activity versus temperature.

4.11 Purification of Alcohol dehydrogenase by Gel filtration Chromatography

Principle of the method. Gel filtration chromatography is a technique for separating substances based on differences in their molecular weight (particle size). Sephadex gels (G-10, G-15, G-25, G-50, G-75, G-100, G-150, G-200) are used as matrices in Gel chromatography. They are based on dextran—a polysaccharide cross-linked to form a three-dimensional porous network with a high degree of hydrophilicity due to A large number of polar hydroxyl groups on their surface. Sephadex is insoluble in water and stable in acidic, alkaline, and salt solutions. Upon immersion in water, Sephadex swells, acquiring The ability to fractionate various molecules according to their size. Larger particles with higher molecular weights move significantly faster than smaller particles, which become trapped within the Sephadex beads.

Thus, molecular separation (fractionation) is achieved, which is used for:

a) Desalting solutions;

b) separating low-molecular-weight molecules from large ones.

Gel filtration is used to purify enzymes from substrates and Cofactors, or proteins from amino acids and Peptides, etc.

Equipment: a photoelectrocolorimeter with a flow-through cell or Uvicord (LKB, Sweden), a chromatography Column (1x30 cm), a freeze dryer (lyophilizer).

Glassware: beaker; 5 mL pipette; elution fraction collection tubes — 50 pcs.

Reagents: 500 mL of 0.05 M Glycine solution; 100 mL of 96% ethanol; Sephadex G-25 fine. Preparation of working solutions. The glycine solution is prepared by dissolving 1.875 g of glycine in 500 mL of distilled water.

The chromatography column is packed with Sephadex G-25 fine, which has been pre-swollen for 24 h at room temperature in distilled water. Before use, the column is washed with 0.05 M glycine solution.

Construction of the calibration curve. The work utilizes a calibration curve plotted for protein determination by the Lowry method.

Procedure. Wheat seeds are homogenized in a mechanical homogenizer until a uniform mass is obtained. Then, 0.05 M glycine solution is added in a 1:3 ratio (3 mL of solution per 1 g of seeds). To remove intact Cells and nuclei, the homogenate is centrifuged for 10 min at 7000 g. Ethanol is added to the supernatant to a final concentration of 70%, shaken vigorously for 5 min, and the mixture is centrifuged. The precipitate, after dissolving in a small amount of 0.05 M glycine solution, is applied to a column (1 x 30 cm) packed with Sephadex G-25 fine and equilibrated with 0.05 M glycine solution; elution is carried out with the same solution. The optical density of the eluted fractions is measured at 280 nm using a Uvicord system (LKB, Sweden) or a spectrophotometer equipped with a flow-through cell. The fractions with the highest activity and lowest Background are lyophilized. The sample contains 90–95% alcohol dehydrogenase relative to the total protein content. Protein concentration is determined by the biuret reaction or the Lowry assay.

Protein yield and activity are calculated based on total protein and total activity values, taking the initial samples as 100%, while the degree of purification is calculated from specific activity values, taking the initial sample readings as unity.

Review Questions for Laboratory Work

1. What is gel chromatography?

2. What are Sephadexes? Where are they used and what are their properties?

3. What is the underlying mechanism of fraction separation in gel chromatography?

4.12 Detection of NAD in Yeast

Principle of the Method

NAD is found in many Organs and tissues of humans and animals, and is particularly abundant in yeast. This coenzyme is readily extracted from yeast with hot water (as it is thermostable) and can be detected by the formation of a fluorescent complex with acetone. This reaction is characteristic of N-derivatives of nicotinic acid amide and is used to determine methylnicotinamide in urine.

Reagents and Test Material:

1) acetone;

2) NaOH solution - 300 g/L;

3) concentrated Hydrochloric acid;

4) alcoholic phenolphthalein solution - 5 g/L;

5) yeast.

Procedure

Place a 4–5 mm piece of yeast into a test tube, add water to fill 1/3 of the tube, and boil for 20–30 seconds, taking care to prevent liquid from spitting out. Filter 5–10 drops of the resulting extract into an empty test tube, add 3–5 drops of acetone, and 1–2 drops of sodium hydroxide solution. Let the test tube stand for 2 minutes; then add 1 drop of phenolphthalein solution and hydrochloric acid drop by drop until the phenolphthalein is decolorized. Shake the test tube continuously while adding the acid. Place the test tube in a boiling water bath for 2 minutes, then cool it and bring it near a turned-on fluorometer.

The acetone complex of NAD fluoresces with blue light.

Review Questions for the Laboratory Practical

1. What is NAD and what role does it play in Enzymatic Catalysis?

2. What enzymes contain NAD as a component?

3. What phenomenon is the detection of NAD in yeast based on?

4.13 Detection of Cytochrome Oxidase in Muscle tissue

Principle of the Method

Cytochrome oxidase is the terminal enzyme in the electron and proton transport chain. In the presence of oxygen, it is capable of oxidizing not only Cytochromes but also several Other Compounds, notably dimethyl-p-phenylenediamine and α-naphthol. The oxidation of these two latter compounds yields indophenol blue. Although this reaction does not occur naturally in Human and Animal tissues, it is widely used to detect cytochrome oxidase. Taking its name from the initial syllables of the words "naphthol" and "dimethyl," it is known as the NADI reaction, and the reagent containing naphthol and dimethyl-p-phenylenediamine is called the NADI reagent.

Reagents and Test Material:

1) NADI reagent (prepared 1 hour prior to the assay): a 1% aqueous solution of dimethyl-p-phenylenediamine is mixed with an equal volume of a 1% alcoholic solution containing 1.5% α-naphthol and a 1.5% sodium carbonate solution; 2) muscle tissue.

Procedure

A small piece of muscle is ground in a mortar with water. A portion of the resulting suspension is transferred to a test tube and thoroughly boiled. Then, the muscle pieces remaining in the mortar and those from the test tube are separated from excess water by filtration. The filter papers containing the muscle pieces are unfolded. Apply 2-3 drops of NADI reagent to the muscle tissue. The unheated muscle gradually develops a blue-violet color, whereas the heated muscle—where cytochrome c oxidase has been inactivated—shows no color change.

Guidelines for Laboratory Report Preparation

In your protocol, state the working principle and outline the procedure briefly. Draw conclusions regarding The properties of NAD.

Review Questions for the Laboratory Practical

1. Why does the reduction of methylene blue by hydrogen occur earlier than that of riboflavin?

4.14 Detection of Catalase in Blood

Principle of the Method

Oxidative processes can generate hydrogen peroxide, which is broken down into water and molecular oxygen by the enzyme catalase (found in various cells, including Blood Cells).

Reagents and Materials:

1) hydrogen peroxide solution - 30 g/L;

2) blood.

Procedure

Add 10-15 drops of hydrogen peroxide and 3 drops of blood to a test tube. Observe the evolution of oxygen bubbles. When a glowing splint is inserted into the tube, it relights because the released oxygen Supports combustion.

Guidelines for Laboratory Report Preparation

Compare the results based on color intensity and draw conclusions regarding the detection of Oxidative Phosphorylation AND the Uncoupling of Respiration and oxidative phosphorylation in rat Liver Mitochondria.

4.15 Muscle Succinate Dehydrogenase and Competitive Inhibition of Its Activity

Succinate dehydrogenase (EC 1.3.99.2) catalyzes The conversion of succinic acid to fumaric acid. FAD serves as a cofactor for the enzyme. The enzyme is tightly bound to The inner mitochondrial membrane.

Principle of the Method

Succinic acid is used as the oxidizable substrate, and the blue dye 2,6-dichlorophenolindophenol serves as the hydrogen acceptor, being reduced to a colorless leuco form. Competitive inhibition of the enzyme is induced by malonic acid, a structural analogue of succinic acid.

Reagents:

1) dye - 2,6-dichlorophenolindophenol, 0.001 N solution;

2) phosphate buffer, 1/15 M, pH 7.4;

3) NaOH, 0.1 N solution;

4) succinic acid (succinate), 3% solution (0.6 M);

5) malonic acid (malonate), 3% solution.

Procedure

1-2 g of fresh muscle tissue is minced with scissors and ground in a mortar with a small amount of water. The resulting paste is transferred onto a double layer of gauze, rinsed with 25 mL of distilled water, squeezed out, and suspended in 4 mL of water. The suspension is divided into 1 mL aliquots across four test tubes.

The contents of the first test tube are boiled for 12 minutes to inactivate the enzyme. Then, reagents are added to the test tubes according to the following table:

Table 4.6 - Experimental Procedure

After 15 minutes, the disappearance of the blue color is observed exclusively In the second test tube.

Guidelines for Laboratory Report Preparation

The results of the experiment should be presented in a table indicating the enzyme, cofactor, electron donor (in tissue or experiment), electron acceptor (in tissue or experiment), reaction product (coloration), and Conclusion.