ENZYMOLOGY - Univerzita...

ENZYMOLOGY

1. Specificity of enzymes (sacharase, α-amylase)

Dependence of enzyme activity on pH (α-amylase)

Estimation of α-amylase in urine (EPS, Wohlgemuth´s test)

2. Estimation of Michaelis´constant of acid phosphatase

Estimation of catalase activity

3. Estimation of lactate dehydrogenase activity in blood serum

Alanine aminotransferase and aspartate aminotransferase in liver

Estimation of alkaline phosphatase in blood serum

4. Inhibition of succinate dehydrogenase with malonate

Estimation of aldolase activity

Estimation of xanthine oxidase activity

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1

Estimation of Michaelis‘ constant (KM) of acid phosphatase Principle: Phosphatases cleav esters bonds of phosphoric acid and phosphate and a relevant alcohol are arised. A synthetic substrate which is possible for a direct photometric estimation is used for observation the enzyme activity. The reaction will be done with different substrate concentration and the relation of the reaction velocity and substrate concentration will be observed. Graphic expression will be in system of reciprocal values. Solutions: citrate buffer pH 4,96 (approximately 0.01 mol/l) substrate solution (4-nitrophenolate, sodium salt 2.5 mmol/l in citrate bufer) inhibition solution (0.1 mol/l of NaOH – 0.03 mol/l EDTA) emzyme solution (acid phosphatase in Eppendorf) Procedure: Read the whole procedure before you start working! To obtain good results it is necessary to pipette exactly and to keep the time of incubation!

1. Prepare 10 test tubes and mark them.

2. Pipette according to the table citrate buffer at first and then substrate (use the mechanical micropipettes, it is possible to use the same tip, after the finishing the work waste the tip).

Test tube No. 1 2 3 4 5 6 7 8 9 10

Citrate buffer [µl] 450 400 350 300 250 200 150 100 50 −

Substrate [µl] 50 100 150 200 250 300 350 400 450 500

3. Place the set of test tubes into the water bath at 37°C for 5 minut. Meanwhile, bring

the enzyme from the refrigerator, prepare stop watch and the pipette of 50 µl with a clean tip.

4. Enzyme solution must be pipetted in absolutely exact 20 seconds period. Add 50 µl of

the enzyme to the test tube No.1, press the stop watch and let it run during the whole experiment. Than pipette the same volume of the enzyme solution to other test tubes in 20 seconds period. Just take out the test tube from the water bath and after adding the enzyme solution place it back immediately. Put the pipette tip approximately 0,5 cm above the level, it is not advisible to touch the substrate. It is necessary to drop the enzyme solution in the substrate not on the wall of the tube.

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5. Prepare the second test tube and add the enzyme after 20 seconds exactly. Continue in

the same way, the period of adding into tubes must be the same, 20 seconds!!!

6. Prepare the inhibition solution which stops the enzyme reaction. The inhibitor must be added in 10 minutes after adding the enzyme, it means the reaction runs for 10 minutes in each test tube. Add 2 ml of inhibition solution to each test tube and mix. Do not place back the tubes to the water bath than switch off the stop watch.

7. Measure the absorbance of all samples at 405nm against water (blank). Start with tube No. 1 and continue in sequence to tube No. 10. Do not rinsen the cuvettes, only pour them and dry them carefully.

8. Enter the obtained values into the table in the computer. The concentration of basic substrate solution is 2.5 mmol/l. Measured absorbance is proportional to the enzymatic reaction velocity in each tube because reaction velocity means amount of converted substrate in time. In this experiment the result is amount of the arisen product in 10 minutes. The program creats Lineweaver-Burk`s graph, it is dependence of 1/v on 1/[S] and the equation for the line is given.

Calculation: Michaelis` constant is estimated from the equation or from the graph where the value of -1/KM is found.

Elaborate a report and add the table and the graph.

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Estimation of catalase activity Principle: Catalase of erythrocytes is a part of a system reducing unadvisable reactive forms of oxygen, more precisely their products. Catalase released from erythrocytes decomposes hydrogen peroxide. Concentration of substrate, i.e. hydrogen peroxide, is determined by manganometric titration. Solutions: substrate solution (0.01 mol/l of hydrogen peroxide in 0.1 mol/l phosphate

buffer pH 6,8) standard solution of potassium permanganate KMnO4 (0.32 g KMnO4 in 1l) sulfuric acid (1 mol/l) enzyme solution (hemolyzed red blood cells 100x diluted) Procedure:

1. Prepare 10 titration flasks, mark them numbers 0-9. Put 2.5 ml of sulfuric acid into each flask (it will stop the enzymatic reaction and it is also necessary for the manganometric titration).

2. Setting up enzymatic reaction:

Measure 60 ml of substrate into 100 ml Erlenmayer flask (buffered hydrogen peroxide solution) and add a magnetic stirrin bar. Place the flask on a magnetic stirrer.

3. Prepare 5 ml pipete for a sampling and transfer 5 ml of substrate into the titration

flask number 0. Be careful, the pipete must not be contaminated with sulfuric acid or else it could not be used for another sampling.

4. Measure 1 ml of enzyme solution (hemolyzed red blood cells 100x diluted) and add it

to the substrate and run the stop watch immediately and let it run.

5. Prepare 5 ml pipete for a sampling in 1 minute intervals and transfer samples into titration flasks immediately. The moment of sample releasing into the titration flask with sulfuric acid that stops the enzymatic reaction is decisive. It means, suck in the sample several seconds before the minute and empty the pipete in a specific moment exactly. All samples are titrated with the solution of permanganate and the concentration of substrate (hydrogen peroxide) in each sample is determined.

6. Repeat the experiment with 10 times diluted enzyme solution, i.e. overall dilution is

1000x (1 ml of enzyme solution + 9 ml of water, or add just 0.1ml of enzyme solution to the substrate).

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7. Values obtained from the titration put in the Excel table in the computer and the expression of changes of substrate concentration and changes of reaction velocity on time.

8. Assess the kinetics and try to estimate a reaction degree.

9. Print the graph and enclose it to your report.

Estimation of lactate dehydrogenase activity in blood serum Principle: Lactate dehydrogenase catalyses the reversible interconversion of pyruvate to lactate with oxidation of NADH + H+ to NAD+. The rate of absorbance decreses of NADH at 340nm is used for kinetic estimation of enzyme activity. Solutions: „working solution“ - 4 ml of R1 (tris 127 mmol/l, pH 7,2, NaCl 323 mmol/l,

pyruvate 2,5 mmol/l) a 1 ml of R2 (NADH 6,6 mmol/l) standard serum examinate serum Procedure:

1. Switch on photometer Libra S 6+ (heating at 37˚C) and adjust the wavelength 340nm.

2. Put a glass cuvette for measuring LD into the photometer.

3. Pipette 1000 µl of destilled water (blank) and measure A = 0.000.

4. Ampty a glass cuvette and put it back into the photometer.

5. Estimation of standard activity: Pipette 20 µl of standard and 1000 µl of „working solution“ ,start up stopwatch, mix the solution and insert it into the cuvette immediately. Measure absorbance at the end of 2nd, 3rd, 4th and 5th minute. Then wash the cuvette with distilled water.

6. Estimation of sample activity: Pipette 20 µl of sample and 1000 µl of „working solution“ ,start up stopwatch, mix the solution and insert it into the cuvette immediately. Measure absorbance at the end of 2nd, 3rd, 4th and 5th minute. Then wash the cuvette with distilled water.

7. Enter the obtained values into a table in the computer (LD, summer semester) and compare the LD value with reference values and evaluate.

Reference value: less than 4,2 µkat/l

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Estimation of ALT and AST activity in liver Principle: The estimation is based on a different light absorption 2,4-dinitrophenylhydrazine of 2-oxoglutarate and pyruvate acid that originates directly for reaction of ALT, after spontaneous decarboxylation of oxalacetate for reaction of AST. The activity of aminotransferases is established from a calibration curve. Solutions: substrate for ALT (DL-alanine, 2-oxoglutarate, phosphate buffer pH 7.4) substarte for AST (L-aspartate, 2-oxoglutarate, phosphate buffer pH 7.4) solution of 2,4-dinitrophenylhydrazine (DNP-hydrazine in HCl) sodium hydroxide NaOH (0,4 mol/l) phosphate buffer pH 7.4 Procedure:

1. Pippete solutions into 4 test tubes according to the table:

Test tubes

1 2 3 4

ALT AST

sample blank sample blank

substrate for ALT 0.50 ml 0.50 ml - -

substrate for AST - - 0.50 ml 0.50 ml 2. Put the test tubes into 37oC water bath for 10 minutes.

3. Preparing of the cell extract:

Crush approximately 1 cm3 of liver tissue in a mortar, add 5 ml of phosphate buffer and continue in homogenisation. All homogenate transfere to a centrifugation tube, centrifugate 5min/2,000 rpm (must do the lab technician). The supernatant is our enzyme solution.

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4. Add enzyme solution into the sample tubes: enzyme solution 0.10 ml - 0.10 ml -

5. Put all 4 test tubes into the water bath at 37oC for 30 minutes and then add the

solution of dinitrophenylhydrazine: dinitrophenylhydrazine 0.50 ml 0.50 ml 0.50 ml 0.50 ml

6. Mix the tubes and let them for 15 minutes at room temperature.

7. Add the solution of sodium hydroxide NaOH into all test tubes:

NaOH 5.00 ml 5.00 ml 5.00 ml 5.00 ml

8. Add enzyme solution into the blank tubes: enzyme solution - 0.10 ml - 0.10 ml

9. Mix the tubes and wait for 5 minutes. Measure absorbance of test tube No. 1 against

No. 2 (it corresponds to the activity of ALT) and absorbance of test tube No. 3 against No. 4 (it corresponds to the activity of AST) at 506nm.

10. Resulting values of activities of ALT and AST are estimated from a calibration curve.

11. Evaluate the results and compare them with reference values.

Reference value: ALT, AST less than 0.7 µkat/l

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Estimation of alkaline phosphatase (ALP) in blood serum Principle: Alkaline phosphatase cleaves substrate 4-nitrophenylphosphate to 4-nitrophenol and inorganic trihydrogenphosphate. Enzyme activity corresponds to the amount of 4-nitrophenol which is estimated photometrically in alkaline environment. Solutions: buffer (methylglucosamine and magnesium sulfate MgSO4)

substrate (4-nitrophenylphosphate + glucose) inhibition solution (EDTA in NaOH) serum (in a small plastic tube - Eppendorf tube)

Procedure:

1. Pipette solutions into two test tubes according to the table: • It is necessary to work very attentively. The serum must be pipetted to the bottom of the tube, it must

not stay on the wall of the tube!

Test tube No. 1

sample Test tube No. 2

blank

buffer 1.00 ml 1.00 ml

serum 0.02 ml - 2. Mix and put both into a water bath at 37oC for 5 minutes:

3. Add substrate to both tubes:

substrate 0.20 ml 0.20 ml

4. Put tubes into a water bath at 37oC for 10 minutes exactly and then stop the reaction by adding the inhibition solution:

inhibitor 0.50 ml 0.50 ml

5. Add just to the tube No. 2 (blank) 0.02 ml of serum:

serum - 0.02 ml

• Both tubes contain the same solutions. The serum was added to the tube No. 2 (blank) after adding the inhibition solution. The reaction can run only in the tube No. 1. The more enzyme is presented in the serum the more substrate is cleaved and the more colored product can be obtained.

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6. Mix the tubes and measure the absorbance (A) of the sample (tube No. 1) against the

blank (tube No. 2) at 420nm. Calculation: Calculation of the catalytic concentration of ALP in an examinated serum.

ALP (µkat/l) = A × 10.236

Evaluate the result and compare with referentce values. Reference value: 0.7 – 2.2 µkat/l (children up to 7.0 µkat/l)

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Inhibition of succinate dehydrogenase with malonate Principle: Succinate dehydrogenase is an enzyme of citrate cycle. It belongs to the riboflavine-linked dehydrogenases presented in the inner mitochondrial membrane. It converts succinate to fumarate under normal conditions. Malonate is known as an competitive inhibitor of this reaction. In the nature conditions this enzyme transfers electrons through the respiratory chain to the oxygen. In order to measure this reaction in our experiment we have to block the respiratory chain, particulary the function of cytochromes with KCN, and we supply an artificial electron acceptor – potassium hexacyanoferrate (III), K 3[Fe(CN)6], which is reduced to potassium hexacyanoferrate (II), K4[Fe(CN)6] . This reduction can be observed photometricaly, and it goes with coloure changing, fading of a yellow solution. In this experiment there is possible to observe the kinetic of enzyme reaction easily even effects of inhibitors. Solutions: pH of solutions is conditioned on 7.2 sodium succinate (0.2 mol/l) potassium cyanide (0,1 mol/l), POISON! potassium hexacyanoferrate (III) (0.01 mol/l) sodium malonate (0,01 mol/l) phosphate buffer pH 7.2 (0.1 mol/l)

a sample of frozen heart for preparing enzyme solution Procedure: 1. Prepare an ice bath. Put several ice cubes and distilled water into 250 ml beaker.

2. Take a calibrated tube and 5x diluted phosphoric buffer, 2 ml of phosphate buffer + 8 ml of

distilled water. Mix it and place into an ice bath.

3. Preparation of the enzyme solution: Take a small piece of pork heart, put it into a mortar and cut it with a scalpel into small pieces. Wash the pieces with approximately 3 ml of diluted phosphate buffer and homogenize the tissue with a pestle. Transfer the crude homogenate into the centrifugation tube and spin it down for 5 minutes at 2,000rpm. Meanwhile, clean the mortar and pestle.

During the preparation of the enzyme solution, it is desirable to prepare a „basic solution“ (point 5 in this procedure). 4. After centrifugation pour the supernatant carefuly in the waste. Put the sediment that must be

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colourless, into the mortar and resuspend it. (Any colour would interfere with photometric measurements.) Add approximately 3 ml of diluted phosphate buffer and glass pieces for better homogenisation (use protective glasses!). Transfer the mixture into the centrifugation tube and spin it down for 5 minutes at 2,000 rpm. After that collect the supernatant because it is our enzyme solution. Store it in the ice bath because mitochondria is very sensitive compages.

5. Preparing of a „basic solution“: Mix 3 ml of potassium cyanide, KCN, 3 ml of potassium hexacyanoferrate (III), K3[Fe(CN)6], and 10 ml of phosphate buffer (undiluted). 6. Pipette the solutions into test tubes according to the table:

Test tube 1 2 3 blank „basic solution“ (ml) 2.0 ml 2.0 ml 2.0 ml - sodium succinate (ml) 1.0 ml 1.0 ml - - sodium malonate (ml) - 0.5 ml 0.5 ml - distiled water (ml) 0.5 ml - 1.0 ml 3.5 ml

7. Switch on the photometer, adjust the wavelength 415nm, and prepare 4 photometric cuvettes, stop

watch, and 0.5 ml pipette. 8. Add 0.5 ml of enzyme solution to the blank, mix and pour it into the cuvette and adjust A = 0.000. 9. Add 0.5 ml of enzyme solution into the test tube No. 1, mix and pour it into the cuvette immediately

and measure absorbance at time 0 and start to measure the time. Note the value of absorbance. 10. Pipette 0.5 ml of enzyme solution into the test tube No. 2 very quickly, mix and pour it into another

cuvette. Place the cuvette into the photometer and measure the absorbance. The time of measurement must be 30 seconds after the measurement of the test tube No. 1 accurately.

11. Change the cuvettes in a period of 30 seconds. Each cuvette is measured in a period of 1 minute.

Make 20 measurements for each cuvette overall. 12. Process the test tube No. 3 which is chacking in the same way. It contains just an inhibitor, no

substrate. Add 0.5 ml of enzyme solution and measure the absorbance in a period of 1 minute. There can be observed just slight decrease of the absorbance what is a systhematical error of the method.

13. Write down all absorbance values in the table into the computer and a graphic processing will be

done. 14. Evaluate the results and make a conclusion.

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Estimation of aldolase activity Principle: Aldolase is a glycolytic enzyme catalysing the reversible conversion of fructose-1,6-bisphosphate to glyceraldehyde phosphate and dihydroxyacetone phosphate. Both products can be evidenced by their reaction with dinitrophenylhydrazine in alkaline media. Hemolysis influences results due to washing out of enzyme from RBCs.

Solutions: solution of fructose-1,6-bisphosphate (0.02 mol/l, pH 7.4)

solution of monoiodoacetic acid (0.002 mol/l, with NaOH, pH = 7,4) solution of hydrazine sulfate (pH = 7.4) collidine buffer (2,4,6-trimethylpyridine, pH = 7.4) solution of dinitrophenylhydrazine in 2M HCl trichloracetic acid solution of sodium hydroxide, NaOH (3%) serum hemolytic serum Procedure:

1. Pipette solutions into 4 centrifugation tubes according to the table:

Test tube No. 1


blank Test tube No. 3


blank

collidine buffer 0.2 ml 0.2 ml 0.2 ml 0.2 ml

serum 0.2 ml 0.2 ml - -

hemolytic serum - - 0.2 ml 0.2 ml

hydrazine sulfate 0.05 ml 0.05 ml 0.05 ml 0.05 ml

monoiodoacetic acid 0.05 ml 0.05 ml 0.05 ml 0.05 ml

trichloracetic acid - 0,4 ml - 0,4 ml

2. Mix the tubes and place them into the water bath at 37°C for 5 minutes.

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3. Add to all tubes 0.1 ml of the solution of fructose-1,6-bisphosphate, mix and place into the water bath at 37°C for 20 min. The reaction does not proceed in test tubes No. 2 and 4 due to trichloracetic acid.

4. Add 0.4 ml of trichloracetic acid even into remaining test tubes No. 1 and 3. An arisen precipitate is cleared off by centrifugating at 2,500 rpm for 10 minutes.

5. Prepare 4 new thin-walled test tubes, mark them in the same way as the centrifugation tubes. Transfer 0.5 ml of clear supernatant to new clean tubes.

6. Add to all test tubes 0.5 ml of 3% solution of NaOH and let the mixtures for 5 min at room temperature.

7. Add to all test tubes 0.5 ml of dinitrophenylhydrazine and mix properly. Place the tubes into the water bath at 37°C for 5 minutes and then add 3 ml of 3% NaOH and let samples for 5 minutes at room temperature.

8. Intesity of coloring is measured, the samples against blanks, i.e. No. 1 against No. 2 and No. 3 against No. 4 in 1 cm cuvettes at 540nm.

Estimation of aldolase activity:

[µkat/l] = A x 0,98 Evaluate your results and compare aldolase aktivity in serum and in hemolytic serum. Reference value: less then 50 nkat/l

Estimation of xanthine oxidase activity Principle: Xanhtine oxidase is a complex molybdenum containing flavoproteine. It is able to convert hypoxanthine to xanthine, and xanthine to uric acid. It is present in many organs and also in milk. Its substrate specificity is not absolute and therfore even non-specific substrates can be oxidated by this enzyme, if a suitable electron acceptor is present. This acceptor is usually the molecular oxygen which subsequently forms water. In this reaction also the one-electron reduction product is often formed, i.e. the superoxide anion O2

- which has to be disproportionated by superoxide dismutase and catalase. In our experiment formaldehyde as a substrate for the xanthine oxidase and the methylen blue as the first electron acceptor are used. The final electron acceptor is the atmospheric oxygen.

This experiment can be used as a simple test for milk pasteurisation. Higher temperature causes enzyme denaturation and adding of formaldehyde cannot raise discolouration of methylen blue. Solutions: solution of methylen blue, 0,01% solution of formaldehyde, 2,0% fresh milk (non-pasteurised) Procedure:

1. Pipette solutions into 4 test tubes according to the table: Test tube No. 1 2 3 4

Fresh milk 5 ml 5 ml 5 ml 5 ml Boil test tubes No. 3 and No. 4 above a burner carefully. Next continue according to

the table: Methylen blue 5 drops 5 drops 5 drops 5 drops Formaldehyde 5 drops - 5 drops -

2. Shake well the tubes and place them into the water bath at 37º C. Look where

discolouration can be observed and explain the reason.

3. Take out the test tube where the blue colour disappeared and shake vigorously until the blue colour is restored. Then continue the reaction in the water bath.

4. You can repeat this cycle several times until all formaldehyde is exhausted. Then

methylen blue will not loose the colour any more. In such a case few drops of additional formaldehyde will restore this ability again.

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