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Laboratory Exercises in Medical Biochemistry
2nd year, General Medicine
WINTER SEMESTER
2019/2020
Department of Medical Chemistry and Biochemistry
Faculty of Medicine in Pilsen, Charles University
Rules of occupational safety
1. Only practising students, specified by the timetable, are right of entry at the practical classes.
No admittance of any visitors. Authorized personnel only.
2. Students are required to familiarize with their task. Laboratory coats and working instructions are
obligatory. Long hair must be adapted for working with a burner without any risk of injury.
Overgarments and bags must be put on the given place.
3. Any leaving is allowed just with a lecturer´s permission.
4. Only prescribed activities are allowed in laboratories. No eating, no drinking, no smoking and no storing
food in laboratories. Laboratory equipment is not allowed to use for any other purposes.
5. If there is a leakage of harmful chemicals possible, the extraction must be ensured. Working with fuming
substances, substances irritating to the respiratory, toxic gases and vapours, as well as annealing and
combustion is allowed to do just in a fume cupboard.
6. Students must be careful during the manipulation with a safety bulb pipette filler. Pieces of broken glass
must be put in a specific container, label "GLASS".
7. It is possible to pour out only the solvents perfectly miscible with water into the sink. They must be
sufficiently diluted (at least 1:10), maximum of 0.5 litre. Aqueous solutions of acids and alkalis must be
diluted at least 1:30. Solvents immiscible with water, poisons, acids and alkalis over the given
concentration and substances loosing toxic gases and gases irritating to the respiratory must be disposed
into the special waste container.
8. An acid is pouring into the water during the dilution of acids, never vice versa.
9. It is forbidden to suck in solution into a pipette per mouth. A safety bulb pipette filler must be used.
10. Spilt acids must be washed by water immediately, if need be neutralized by sodium carbonate. Spilt
alkalis must be just washed by water.
11. All burners and electrical current must be switched off due to spilling of flammable liquids and it is
necessary to clear the air. Pouring liquids must be absorbed by suitable porous material and it is liquidate
in the appropriate way.
12. During the heating of a liquid in a boiling flask superheating must be prevented by using a boiling chip.
13. It is necessary to check all devices before the start of working. Possible faults and defects must be
reported to a lecturer or a laboratory technician.
14. Intentional handling with electrical device and substances is forbidden. To switch on a device and to light
a burner is allowed by the approval of a lecturer or a laboratory technician.
15. All centrifugation procedures must be controlled by a lecturer or a laboratory technician. Vessels for the
centrifugation must be well balanced and the top of the centrifuge must be closed safely during the
operation.
16. The gas intake and electrical current must be switch off and clear the air if there is a leakage of gaseous
fuels.
17. A lighted burner without supervision is not allowable. If there are any problems with a turner, it is
necessary to switch off the gas intake and the burner must be regulated.
18. Students are obliged to inform a lecturer of any accident, injury, or in case of ingestion chemicals.
19. Serious breach of rules because of a lack of discipline or ignorance is the reason of leaving the practical
classes as an unexcused absence.
20. Students must be informed about classification of toxic, carcinogenic, mutagenic and damaging fertility
substances. Safety sheets of particular substances are available in laboratories.
21. Students must be informed about rules of occupational safety with highly toxic substances (label T+)
using in laboratories (e.g. mercury, potassium cyanide, ethidium bromide, mercury (II) nitrate).
Pilsen, September 16th, 2016 Ing. Václav Babuška, Ph.D.
Deputy Head of the Department
LIST OF EXERCISES
Pipetting training: page 4
Pipetting of distilled water with checking of the precision by weighting
Determination of the density of an unknown solution
Preparation of a solution and its aliquoting into individual tubes
Pipetting of large volumes
Optical methods: page 6
Spectrophotometric estimation of Cu2+ concentration
Identification of acid-base indicator by absorption spectra
Spectrophotometric estimation of Cl- concentration
Chromatography: page 12
Paper chromatography of amino acids
Separation of plant pigments by thin-layer chromatography
Separation of dye mixture by gel chromatography
Potentiometry: page 19
Potentiometric measurement of pH
Demonstration of buffer functioning
Potentiometric titration - estimation of HCl concentration
- estimation of CH3COOH concentration
Determination of the isoelectric point of an amino acid
Osmolality, osmosis, dialysis: page 27
Demonstration of osmosis
Determination of osmolality using cryoscopy
Monitoring the kinetics of dialysis
Antioxidants: page 35
Measurement of total antioxidant capacity (TAC)
Enzymology I: page 40
Enzyme specificity
Dependence of activity of α-amylase on pH
Estimation of alpha-amylase in urine (EPS, Wohlgemuth´s test)
Enzymology II: page 46
Estimation of Michaelis' constant of acid phosphatase
Estimation of catalase activity
Enzymology III: page 50
Estimation of lactate dehydrogenase activity in blood serum
Alanine aminotransferase and aspartate aminotransferase in liver
Estimation of alkaline phosphatase in blood serum
Enzymology IV: page 55
Inhibition of succinate dehydrogenase with malonate
Estimation of aldolase activity
Estimation of xanthine oxidase activity
4
Pipetting training
1) Pipetting of distilled water with checking of the precision by weighting
a) Pipetting of large volumes, pipettes with fixed volume
Place a small empty beaker on a balance pan. The mass of the empty vessel is called the
tare. Press the TARE button to get a reading of 0.000 g.
Take the beaker from the balance, place it on the table and pipette into it distilled water of
the following volumes:
3 x 2000 l
2 x 500 l
For pipetting of 2 ml (=2000 l), use the pipette with the fixed volume. Select the proper tip for the pipette
(big white). To attach the tip, firmly press the shaft of the pipette into the large open end of the tip with light
force to ensure a good seal. Similarly, for pipetting of 0.5 ml (=500 l), use the pipette with the fixed
volume and proper tip.
After you finish the pipetting, place the beaker on a balance pan to read the weight of
distilled water inside. Compare the result with a theoretical expected value obtained as
a sum of pipetted volumes recalculated to mass using the density. For doing this, density
of distilled water at the temparature in the laboratory is =1.000 g/cm3.
Expected volume Expected mass Measured mass
b) Pipetting of small volumes, pipettes with adjustable volume
Place an empty small plastic test tube of a volume 1.5 ml, so called Eppendorf tube, on a
balance pan, and press the TARE button.
Take the open Eppendorf tube in your hand and pipette into it distilled water of the
following volumes:
375 l
25 l
For pipetting of 375 l, use the pipette with adjustable volume in the range 100-1000 l and proper tip.
For pipetting of 25 l, use the pipette with adjustable volume in the range 20-200 l and proper tip (yellow).
After you finish the pipetting, close the Eppendorf tube and place it on a balance pan to
read the weight of distilled water inside. Compare the result with a theoretical expected
value obtained as a sum of pipetted volumes recalculated to mass using the density. For
doing this, density of distilled water at the temparature in the laboratory is =1.000 g/cm3.
Expected volume Expected mass Measured mass
5
2) Determination of the density of an unknown solution
Place an Eppendorf tube on a balance pan and press the TARE button.
Into the Eppendorf tube, pipette exactly 1.000 ml (=1000 l) of a solution, density of
which you want to determine.
For pipetting of 1000 l, use the pipette with adjustable volume in the range 100-1000 l and proper tip.
Close the Eppendorf tube and place it on a balance pan to read the weight of a solution
inside. From a known volume and mass measured, calculate the density.
Volume Mass Density
3) Preparation of a solution and its aliquoting into individual tubes
Pipette into an Eppendorf tube: distilled water 93 l
dye solution 7 l
For pipetting of 93 l, use the pipette with adjustable volume in the range 20-200 l and proper tip (yellow).
For pipetting of 7 l, use the pipette with adjustable volume in the range 0.5-10 l and proper tip (very small
white). At this step, you are adding a very small volume. The best way how to do it: The orifice of the tip
must be dipped below the level of the solution which is already in the Eppendorf tube. By adding these 7 l,
you will simultaneously mix the solution, read further how to do this!
Mix thoroughly the content of the Eppendorf tube. It can be done by so called "pipetting
up and down" several times, i.e. repeatedly pressing and releasing the button of the pipette
causing movement of the solution in the tip "up and down". In this exercise, the solution is
coloured, so you can see what is happening and check if mixed sufficiently. Taking the tip
out of the Eppendorf tube, be careful to make the tip empty!
Prepare 5 microtubes (0.2 ml) in a rack and pipette into each exactly 20 l of the solution
you have prepared.
5 x 20 l
100 l
Evaluate the precision and accuracy of your pipetting!
4) Pipetting of large volumes
Transfer into titration flask: distilled water 10.0 ml
0,1 M HCl 5.0 ml
indicator (methyl red) 5 drops
For pipetting of 10.0 ml distilled water, use the glass pipette and rubber suction bulb. 5 ml of 0.1 M HCl add
from dispenser. "up and down". Adding of 5 drops of indicator as the last step.
6
Optical methods
1) Spectrophotometric estimation of Cu2+ concentration
The hydrated copper complex ion [Cu(H2O)4]2+ is light blue in colour. However, for photometric
estimation, this coloration is of low intensity and it must be highlighted with a suitable reagent like solution of
ammonia that results in formation of dark blue tetraammine copper(II) complex.
[Cu(H2O)4]2+ + 4 NH3 [Cu(NH3)4]2+ + 4 H2O
For determination of the Cu2+ concentration, we will use a calibration curve method. By diluting of
a stock solution (a standard solution of known high concentration) you will get a set of calibration solutions.
Measure absorbances of them at optimal wavelength (absorption spectrum maximum). The calibration curve is
a graph of absorbance as a function of concentration, A = f(c). According to the Beer-Lambert law, it should be
a straight line passing through the origin, i.e. the absorbance (A) is directly proportional to the concentration (c).
Once having a calibration curve, concentration of unknown samples can be easily read from it.
Procedure:
A) Prepare the solutions
Into a test tube rack, place clean and dry test tubes and mark them with numbers:
1 to 10 calibration solutions
0 blank solution
S1, S2, S3, S4 unknown samples
According to the table, prepare the calibration solutions by diluting of the stock solution
of Cu2+ (c = 25 mmol/l) with distilled water. Be careful with pipetting accuracy!
Number Stock solution H2O c(Cu2+)
- ml ml mmol/l
0 - 5.0
1 0.5 4.5
2 1.0 4.0
3 1.5 3.5
4 2.0 3.0
5 2.5 2.5
6 3.0 2.0
7 3.5 1.5
8 4.0 1.0
9 4.5 0.5
10 5.0 -
Transfer by pipetting 5.0 ml of each unknown sample solution into the test tubes marked
S1-S4.
Add 5 ml of ammonia solution into each test tube (blank, calibration solutions, unknown
samples). For doing this, use the automatic dispenser.
Mix the content of all test tubes by turning upside down and back. For closing the tubes,
use the rubber stopper not your thumb!
7
B) Make a search for the optimal wavelength
On a spectrophotometer, measure the absorbance of the calibration solution with the mean
concentration (number 5) against the blank at wavelengths specified in the table below.
Pour appropriate volume of calibration solution 5 into a clean cuvette, similarly fill the
other cuvette with the blank solution. Be careful inserting the cuvettes into the
spectrophotometer not to spill the content!
Wavelength [nm] 400 450 500 550 600 650 700
Absorbance
Select the optimal wavelength for making the further measurements. Optimal wavelength
is the wavelength with the highest absorbance.
Optimal wavelength [nm]
C) Construct the calibration curve
Before plotting the calibration curve, you have to calculate the concentration of individual
calibration solutions from known dilution of the stock solution.
On a spectrophotometer, measure the absorbances of all the calibration solutions against
the blank at the optimal wavelength found in previous step.
Number Concentration Absorbance
- mmol/l -
0 0.0 0.000
1
2
3
4
5
6
7
8
9
10
According to the Beer-Lambert law, the absorbance is directly proportional to the
concentration. Therefore the calibration curve should be a straight line passing through the
origin, i.e. the point (0,0).
There is a computer in the students' laboratory with a MS Excel file prepared to fit a linear
regression line to the collected data. Before putting data into this MS Excel form, please
finish the measurement of unknown samples (next step).
8
D) Determination of concentration of unknown samples
On a spectrophotometer, measure the absorbances of all unknown sample solutions
against the blank at the optimal wavelength.
Sample Absorbance Concentration
- mmol/l
1
2
3
4
The regression equation for calibration curve generated in the previous step will be
automatically used to calculate concentrations of unknown samples.
Print the filled MS Excel form and attach it to your lab report.
Conclusion:
9
2) Identification of acid-base indicator by absorption spectra
Absorption spectrum is a graph of absorbance as a function of wavelength of light (electromagnetic
radiation). In UV-VIS range of radiation, absorption spectrum looks like a continuous curve with one or only
few maxima, whose position is characteristic for particular substance. Absorption spectra can be used for
identifying substances or for checking their purity.
Procedure:
Acid-base indicators are mostly organic dyes, which can be protonated (change ionization) according to pH
of a solution into which they are introduced. A change in ionization is coupled with a change in structure and
coloration. Acidic form and basic form of an indicator are different in colour. For identifying of an unknown
acid-base indicator, we will measure absorption spectra of both acidic and basic form.
You will get a test tube with an unknown acid-base indicator to identify. It is an aqueous
solution at pH more-less neutral (pH~7).
What is the colour of the indicator at neutral pH?
Take 2 clean test tubes. Mark one "A" (acidic) and the other one "B" (basic). Measure 1
ml of the indicator solution into both test tubes and then add 1 ml of sulfuric acid (0.05
mol/l) to "A" and 1 ml of sodium tetraborate (0.05 mol/l) to "B".
What is the colour of the acidic form?
What is the colour of the basic form?
On a spectrophotometer, measure the absorption spectra within the range 400 - 700 nm
using the interval of 10 nm of both acidic and basic forms of the indicator against distilled
water as a blank.
There is a computer in the students' laboratory with a MS Excel file prepared to put the
collected data and plot the graphs.
Print the filled MS Excel form and attach it to your lab report.
Compare the spectra with standards (from a catalogue available in the students'
laboratory) and identify the indicator.
10
Wavelength
[nm]
Absorbance
(acidic form)
Absorbance
(basic form) Wavelength
[nm]
Absorbance
(acidic form)
Absorbance
(basic form)
400
560
410
570
420
580
430
590
440
600
450
610
460
620
470
630
480
640
490
650
500
660
510
670
520
680
530
690
540
700
550
Wavelength of the absorption maximum of the acidic form:
Wavelength of the absorption maximum of the basic form:
Name of unknown acid-base indicator:
Try to find somewhere the structural formula:
11
3) Spectrophotometric estimation of Cl- concentration
Reagent for estimation of Cl- concentration contains Hg(SCN)2 and Fe(NO3)3. Cl- reacts with Hg(SCN)2
when the colorless complex [HgCl2] is formed. Free ions SCN- yields with Fe3+ red complex suitable for the
photometric determination.
Procedure:
You have prepared 3 small dry test tubes into a test tube rack. Mark them with sa
(sample), st (standard) and 0 (blank). Pipette according to the table blood serum,
standard and distilled water (on the bottom of test tube, the solution mustn´t stay
inside the tip!):
Test tube 1 (sample) 20 µl blood serum
Test tube 2 (standard) 20 µl standard Cl- (cst = 100 mmol/l)
Test tube 3 (blank) 20 µl distilled water
Pipette 2.0 ml of reagent into all test tubes. Mix the content properly.
Mix and after 5 minutes measure the absorbance of sample (Asa) and standard (Ast)
against blank at 450 nm.
Absorbance
Sample
Standard
The concentration of Cl- calculate according to: x cstandard
Csample = mmol/l
12
Chromatography
1) Paper chromatography of amino acids
1. Take a sheet of chromatographic paper (18 x 18 cm). Find out a centre of the sheet. Draw
a circle around the centre about 3 cm in diameter. You can use prepared template. This is
the "base-line", the start position. On the base-line make 6 marks evenly spaced and
number them 1-6.
Chromatogram
2. Using labelled capillaries make small spots (less than 0.5 cm in diameter) of about 5 μl of
the standards and your unknown sample. The application should be repeated 2 times.
Before the further application, the wet spots must be dried under the infra-red lamp or
a ventilator.
3. Use your pencil to perforate the paper exactly in the centre to draw through the hole
a paper "wick". Place the paper into a Petri dish partly filled with a solvent mixture
(n-butanol, acetic acid, water – 4:1:1) and cover the paper with a cap. Do not take the
Petri dish out from the fume chamber! All following procedures must be done in a fume
chamber!
Petri dish with solvent chromatogram paper wick
13
4. The chromatograms are left to develop until the head of a solvent overlaps the Petri dish
cap. The paper is removed, dried, and sprayed with ninhydrine reagent. Heat the paper at
80°C in order to develop the formation of blue complexes.
5. Calculate the individual Rf according to the equation.
Site Amino acid Rf
1 Lysine
2 Glycine
3 Alanine
4 Isoleucine
5 The mixture of 1-4 - - -
6 Unknown sample
Sample identification:
14
2) Separation of plant pigments by thin-layer chromatography
There are a few lipophilic pigments in green parts of plants. They have different chemical and
functional properties. The primary function of pigments in plants is photosynthesis which
uses the green pigment chlorophyll along with several red and yellow pigments. These
pigments can be separated using Thin Layer Chromatography (TLC).
Procedure:
1. There are prepared samples of green leaves or needles on the working place. Cut it in
pieces in a mortar. Then add a small spoon of calcium carbonate, a little bit of glass
shards and crush it with a pestle in the presence of 1-2 ml of acetone. Protect your
eyes using glasses.
2. Take two TLC plates. After the separation, one is for you to be attached to the
protocol, the second will be used further and destroyed by procedure of extraction of
one of the pigments.
3. Draw the base-line about 2 cm from the shorter edge of the plate. Use the thin pencil.
4. Dip the glass capillary into the acetone extract and touch the tip of the capillary to the
plate at the position you’ve marked. Gently and quickly make spot according to the
picture. Try to make as small spots as possible and avoid scratching of the thin layer.
For higher concentration repeat the process of application for several times.
5. Prepared separation chamber should be kept close. There is approximately 1cm thick
layer of liquid solvent mixture on the bottom and the rest volume is filled with its
vapors.
6. Place the thin layer plate carefully in the chamber (chromatography tank), containing a
solvent at the bottom. Cover the vessel with a glass plate and allow to separate about
15 min. The base – line must be situated above the level of a solvent.
15
7. When the front of a solvent almost overlaps the top of the TLC plate, remove the plate.
Evaluate the dry plate for the presence of pigments and enclose to laboratory protocol.
16
Measurement of absorption spectrum of one of the pigments
After separation of the plant pigments, take one of the TLC plates and select one of the green
strips. Use the scissors to cut out the strip from the TLC plate. Put the piece with the strip into
a test tube and add 2 ml of ethanol. Wait until complete discoloration of TLC plate piece.
Measure absorption spectrum of this fraction using a spectrophotometer in a range of
wavelengths from 350 to 700 nm. As a blank solution, use distilled water. By comparing the
spectrum with spectra of individual pigments, determine whether the fraction contains
chlorophyll a or chlorophyll b.
wavelength
nm A
wavelength
nm A
wavelength
nm A
350 470 590
360 480 600
370 490 610
380 500 620
390 510 630
400 520 640
410 530 650
420 540 660
430 550 670
440 560 680
450 570 690
460 580 700
Name of the pigment
17
3) Separation of dye mixture by gel chromatography
Task: Separate dextran blue and potassium chromate.
Dextran blue
What are dextrans?
Colour Molar mass
~ 2 × 106 g/mol
Potassium chromate
formula:
Colour Molar mass
g/mol
There is a chromatographic column filled with Sephadex gel on the working place. Sephadex
is polysaccharide of dextran type.
Procedure:
(1) Uncap the chromatographic column, open the tap and allow carefully run out the
excess of fluid until the surface of the gel bed is reached. Pipette on the gel slowly 0.5
ml of the coloured mixture (potassium chromate and dextran blue). Use the pipette
with fixed volume (=500 µl) and proper tip (blue). Pipette carefully! Not to whirl the
surface of the gel!
Colour of the mixture:
(2) By turning the tap let the sample pass into the column.
(3) Pipette (using glass pipette and rubber suction bulb) about 3 ml of elution solution
(physiologic solution; 0.9% NaCl) and let it penetrate the gel in the same way.
(4) Elute the column with sufficient amount of elution solution until both fractions leave
the outflow.
(5) Collect both fractions in calibration test tubes.
18
What is happening in the columne? Describe the changes:
Fraction
order Colour Substance Fraction volume
1
2
Explain the order of separated dyes:
19
Potentiometry
1) Potentiometric measurement of pH
Determination of pH can be done by simple colorimetric methods using acid-base indicators
(pH test strips). Nevertheless, the precision of such methods is mostly insufficient. For exact
pH measurement, laboratories are equipped with pH meters with usual resolution of 0.01 pH
units, high-end instruments with resolution of 0.001 pH units.
There is a pH meter with combined electrode (consisting of both the glass and the reference
electrode) at your working place. Electrode must be kept moist using the storage solution.
Remove the protective plastic cover with storage solution before using the electrode. Keep the
storage solution inside the plastic cover, do not pour it out. At the end of your work, you have
to place the electrode back into the cover with storage solution. Never touch the glass
membrane of the electrode with your fingers! Electrodes should be rinsed between samples
with distilled water. After rinsing, gently blot the electrode with cotton paper piece to remove
excess water.
Procedure:
Into a clean small beaker, transfer the sample which pH you want to determine, by
pouring from the small plastic bottle.
Estimate the pH using universal indicator strip.
Dip the electrode of the pH meter into the solution in the beaker, gently shake the content
by slowly moving the beaker to make the membrane of the electrod be in contact with the
solution. (Electrode is "dirty" with distilled water used for rinsing between samples.)
After stabilization of the value, read the pH on a display.
Put the sample back into the small plastic bottle.
Sample pH estimated by indicator strip pH measured by pH meter
20
2) Demonstration of buffer functioning
Buffers are solutions that maintain a relatively stable pH and resist changes in pH, upon
addition of small amounts of acid or base. At your working place, there are two bottles with
solutions to compare how they are resistant to changes in pH. One bottle contains unbuffered
solution - deionized (ultrapure) water (in theory, pH=7), the other one contains phosphate
buffer at pH close to 7.
Unbuffered solution - water
Using a graduated cylinder, measure 50 ml of deionized water, pour it into a clean beaker and
determine the pH by pH meter. Record the value (measurement 1). Pour out the water from
the beaker. Using the same graduated cylinder, measure again 50 ml of deionized water, pour
it into the same beaker and determine the pH by pH meter (measurement 2). After the second
measurement, do not remove the combined electrode from the beaker, let it dipped there. By
pipetting add 100 μl of HCl solution (c=0.1 mol/l) into the solution in the beaker and gently
mix the content. After stabilization of the value record the pH.
pH of deionized water pH after addition of HCl
measurement 1
measurement 2
Describe how stable is the pH of an unbuffered solution:
Buffer
Using a graduated cylinder, measure 50 ml of phosphate buffer, pour it into a clean beaker
and determine the pH by pH meter. Record the value (measurement 1). Pour out the buffer
from the beaker. Using the same graduated cylinder, measure again 50 ml of phosphate
buffer, pour it into the same beaker and determine the pH by pH meter (measurement 2).
After the second measurement, do not remove the combined electrode from the beaker, let it
dipped there. By pipetting add 100 μl of HCl solution (c=0.1 mol/l) into the solution in the
beaker and gently mix the content. After stabilization of the value record the pH.
pH of the buffer pH after addition of HCl
measurement 1
measurement 2
Describe how stable is the pH of a buffer solution:
Describe the differences between the behaviour of the buffer solution, and the pure water,
upon the addition of a small amount of a strong acid:
21
3) Potentiometric titration
Potentiometric titration is a volumetric method in which the potential between two electrodes
is measured as a function of the added reagent volume. In potentiometric titrations, there is no
need for indicator. The titration is not stopped at the equivalence point, the whole titration
curve is constructed instead. Volume of the standard reagent consumed to reach the
equivalence point is found by analysis of the titration curve. A titration curve has a
characteristic sigmoid shape. The part of the curve that has the maximum change marks the
equivalence point of the titration.
You will be performing two potentiometric titrations to determine the unknown
concentrations of two acids. In order to perform these titrations you will utilize a pH meter.
A) Strong acid: hydrochloric acid (HCl)
B) Weak acid: acetic acid (CH3COOH)
A) Titration of a strong acid, graphical analysis of the titration curve
Procedure:
Into a clean small beaker, using a glass pipette measure exactly 10.0 ml of the sample
(strong acid – HCl) which concentration you want to determine. Add a small stirring bar
(little white corpuscle similar to Tic Tac mints) into the beaker.
Close the burette tap, and fill the burette with the standard solution of NaOH (c=0.100
mol/l) until the meniscus is about 1-2 cm above the zero mark. Remove the funnel. Open
the tap and allow the solution to drain (into the waste bottle) until the meniscus falls to
zero mark. Fill the burette with the standard solution. The burette is ready for the titration.
Set up the apparatus so that you can perform the potentiomeric titration. Place the beaker
with the sample on a magnetic stirrer. Dip the electrode of the pH meter into the solution
in the beaker. If the electrode is not sufficiently submerged, add a little of distilled water.
Place the burette so that the orifice is slightly above the beaker and is possible to use it for
adding standard solution to the sample. Switch on the stirring (not heating!) and adjust the
speed of it to optimal (not extremely fast to prevent damage of the electrode).
Before adding any NaOH from the burette, record the initial pH on the display of the
pH meter. Put the data into the table further in this instruction sheet. You are now ready to
begin the titration. You will be adding NaOH solution in increments 0.5 ml until 10.0 ml.
After every addition of NAOH, record the pH. Be sure that the pH has stabilized! There is
a delay between the addition of NaOH and a stable pH reading.
There is a computer in the students' laboratory with a MS Excel file prepared to put the
collected data and plot the graph (titration curve).
Print the filled MS Excel form.
Use a graphical method to find out the equivalence point.
Two parallel lines are drawn tangent to the flat
portions of the curve. A line perpendicular to both the upper and the
lower tangent is drawn to help you find the middle between them so that
you can plot the third parallel line exactly in the middle. The point where
this middle parallel crosses the titration curve is the equivalence point.
22
Potentiometric titration of a strong acid (HCl) – data sheet
Added NaOH volume (ml) pH
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
HCl
Consumption of NaOH read from titration curve:
Calculation: (M = 36.5 g/mol)
Molar (substance) concentration c = mmol/l
Mass concentration = g/l
23
B) Titration of a weak acid, graphical + methematical analysis of the titration curve
Procedure: (the same as for strong acid)
Into a clean small beaker, using a glass pipette measure exactly 10.0 ml of the sample
(weak acid – CH3COOH) which concentration you want to determine. Add a small
stirring bar (little white corpuscle similar to Tic Tac mints) into the beaker.
Close the burette tap, and fill the burette with the standard solution of NaOH (c=0.100
mol/l) until the meniscus is about 1-2 cm above the zero mark. Remove the funnel. Open
the tap and allow the solution to drain (into the waste bottle) until the meniscus falls to
zero mark. Fill the burette with the standard solution. The burette is ready for the titration.
Set up the apparatus so that you can perform the potentiomeric titration. Place the beaker
with the sample on a magnetic stirrer. Dip the electrode of the pH meter into the solution
in the beaker. If the electrode is not sufficiently submerged, add a little of distilled water.
Place the burette so that the orifice is slightly above the beaker and is possible to use it for
adding standard solution to the sample. Switch on the stirring (not heating!) and adjust the
speed of it to optimal (not extremely fast to prevent damage of the electrode).
Before adding any NaOH from the burette, record the initial pH on the display of the
pH meter. Put the data into the table further in this instruction sheet. You are now ready to
begin the titration. You will be adding NaOH solution in increments 0.5 ml until 10.0 ml.
After every addition of NAOH, record the pH. Be sure that the pH has stabilized! There is
a delay between the addition of NaOH and a stable pH reading.
There is a computer in the students' laboratory with a MS Excel file prepared to put the
collected data and plot the graph (titration curve).
Print the filled MS Excel form.
Use both, a graphical method and mathematical analysis to find out the equivalence point.
Mathematical analysis of the titration curve
Consumption of the standard reagent in equivalence point (V) can be calculated using the
formula:
ΔVpHΔpHΔ
pHΔVV
22
2
V+ "added NaOH volume" at last positive 2pH
2pH+ the last positive 2pH
2pH- absolute value of the first negative 2pH
V the difference in "added NaOH volume" between the last positive and the first
negative 2pH (In our experiment, it always was 0.5 ml.)
24
Potentiometric titration of a weak acid (CH3COOH) – data sheet
Added NaOH
volume (ml) pH pH 2pH
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
pH = pHx+1 - pHx 2pH = pHx+1 - pHx
25
CH3COOH
Consumption of NaOH read by graphical method from a titration curve:
Consumption of NaOH calculated:
Calculation: (M = 60.0 g/mol)
Molar (substance) concentration c = mmol/l
Mass concentration = g/l
26
4) Determination of the isoelectric point of an amino acid
Added NaOH
volume (ml) pH
Added NaOH
volume (ml) pH
0.0
- -
0.5
10.5
1.0
11.0
1.5
11.5
2.0
12.0
2.5
12.5
3.0
13.0
3.5
13.5
4.0
14.0
4.5
14.5
5.0
15.0
5.5
15.5
6.0
16.0
6.5
16.5
7.0
17.0
7.5
17.5
8.0
18.0
8.5
18.5
9.0
19.0
9.5
19.5
10.0
20.0
Calculation of pI:
27
Osmolality, osmosis, dialysis
1) Demonstration of osmosis
We will try to carry out a classical experiment on
demonstration of osmosis. The principle is shown in
the figure. Water moves from the solution of lower
osmolality, across the semipermeable membrane,
into the solution of higher osmolality, and its level
rises. In theory, the process should stop in time of
balancing the osmotic pressure by hydrostatic
pressure of the column of the solution inside a tube.
π = h g
h ... difference in solution levels
density of the solution
g ... gravitational acceleration
1. On a wider portion of the tube, fasten the cellophane, which will serve as a semi-
permeable membrane.
2. Dip the tube membrane down into a graduated cylinder with distilled water so that the
water level in the cylinder reached the mark.
3. Into the tube, transfer carefully by pipetting saturated sucrose solution, so that liquid
levels in the tube and in the cylinder are at the same level. For better visibility, sucrose
solution was colored with blue food coloring.
4. Let the experiment run until the end of the class. Then, using a ruler, measure the
difference between the two levels and make a conclusion.
The beginning: "What time is it?"
The end: "What time is it?"
Time period:
min
Difference in solution levels (mm):
Conclusion:
28
29
2) Determination of osmolality using cryoscopy
Preparing of 250 ml of Ringer's solution
Physiological solution in form of 0.9% NaCl contains only Na+ and Cl- ions. However, there
are also other ions in the blood plasma. In clinical practice, there are more types of infusion
solutions in use, some of them more similar in ionic composition to blood plasma. Ringer's
solution is one of them.
1. Weigh stepwise using a plastic weighing boat and transfer weighed quantities into an
Erlenmayer flask (of volume of 250 ml):
sodium chloride (NaCl) 2.150 g
potassium chloride (KCl) 0.075 g
calcium chloride (CaCl2) 0.083 g
2. Into the same Erlenmayer flask, flush also unobservable remnants from the plastic
weighing bottle using a squirt bottle with distilled water.
3. Into the same Erlenmayer flask, add distilled water to a volume about 100 – 150 ml and
by swirl mixing thoroughly dissolve the contents.
4. Using a funnel pour the content of Erlenmayer flask into a 250 ml volumetric flask. Rinse
the Erlenmeyer flask at least 2x with a little of distilled water, pour everything into a
volumetric flask.
5. Fill the volumetric flask exactly to the mark with distilled water (i.e. a volume of 250 ml).
6. Close the volumetric flask by a stopper and thoroughly mix the contents.
What is the concentration of the individual ions in the solution prepared?
M(NaCl) = 58.45 g/mol
M(KCl) = 74.56 g/mol
M(CaCl2 . 2H2O) = 146.99 g/mol
Result
Na+ mmol/l
K+ mmol/l
Ca2+ mmol/l
Cl- mmol/l
From the known composition, calculate the osmolality (osmolarity) of this solution:
30
Measurement of osmolality by modern osmometer
There is a modern freezing point osmometer Osmomat 3000 in the laboratory. The total
osmolality of aqueous solutions is determined by comparative measurements of the freezing
points of pure water and of solutions. The instrument requires very small sample volumes (50
l), test time is short (60 s).
Preparing samples:
Distilled water – it is available in the laboratory
Ringer's solution – you have already prepared (previous exercise)
Ringer's solution + ethanol
Using a graduated cylinder, measure 50 ml of Ringer's solution, pour it into a small beaker
and add by pipetting 0.25 ml of 40% ethanol.
Calculate what rise of osmolality (osmolarity) should the addition of ethanol result in:
Ringer's solution + glucose
Using a graduated cylinder, measure 50 ml of Ringer's solution and pour it into a small
beaker. Weigh using a plastic weighing boat 270 mg of glucose and dissolve it in the solution
in the beaker.
Calculate what rise of osmolality (osmolarity) should the addition of glucose result in:
Sample Measured osmolality
Distilled water mmol/kg
Ringer's solution mmol/kg
Ringer's solution + ethanol mmol/kg
Ringer's solution + glucose mmol/kg
31
3) Monitoring the kinetics of dialysis
This exercise demonstrates separation of high and low molecular weight substances (starch
and NaCl) by dialysis. Moreover, we will monitor dialysis rate of chlorides. As a dialysis membrane,
we will use a cellophane with the size of pores between 3 and 4 nm. These pores are permeable to
small ions, but do not allow the passage of large molecules.
Procedure:
Preparing the dialysis experiment:
1. There is an empty dialysis container mounted the laboratory stand at your working
place. The dialysis container is a glass tube with cellophane membrane on one side.
2. Using a graduated cylinder, put 150 ml of distilled water into the beaker (of a volume
of 250 ml). Keep the dialysis container out of the beaker at this moment. Add
a stirring bar into the beaker, place the beaker on a magnetic stirrer, switch on the
stirring (not heating!) and adjust the speed of it to optimal.
3. Into the dialysis container, pipette 5 ml of starch solution and 10 ml of NaCl
solution (c = 1.5 mol/l). Prepare a stopwatch so that it can be started immediately
when you dip the dialysis container into the beaker (cellophane membrane must be
below the level of the solution in the beaker). Before starting the experiment,
ensure yourself you know what to do further (taking the samples as described
below)! Let the experiment run for 90 minutes.
Monitoring of dialysis rate:
At points in time of 2, 4, 6, 8, 10, 15, 20 minutes after starting the experiment, and
later in 10-minute intervals, take (using a pipette) from the beaker samples of a
volume of 2.5 ml. Put the samples into titration flasks labelled with numbers
corresponding to the time sample was taken.
Time (min) 2 4 6 8 10 15 20
Vtitration (ml)
Vdif (ml)
c (mmol/l)
n (mmol)
Time (min) 30 40 50 60 70 80 90
Vtitration (ml)
Vdif (ml)
c (mmol/l)
n (mmol)
Vtitration mercurimetric titration of the sample - consumption of the standard reagent
Vdif volume of the solution in the beaker (It is getting smaller by taking samples!)
c concentration of Cl- in the solution in the beaker
n amount of Cl- in the solution in the beaker
32
Estimation of chloride concentration:
Concentration of Cl- can be estimated by mercurimetric titration. Mercurimetry
belongs to complexometric titration methods. Standard reagent is a solution of Hg(NO3)2, that
forms with Cl- ions soluble non-dissociated complex [HgCl2]:
Hg2+ + 2 Cl- [HgCl2] f = n(Cl-)/n(Hg2+) = 2/1
For indication of the end point of the titration, we will use diphenylcarbazone that forms
with the excess of Hg2+ ions blue-violet coloration. The solution of Hg(NO3)2 is toxic! Keep
safety in mind! After finishing your work, put the waste into the extra bottle!
To the sample taken from the beaker you put into a titration flask, add 2.5 ml of distilled
water and 0.3 ml of indicator (diphenylcarbazone). Titrate with standard solution of Hg(NO3)2
(cstandard reagent = 12.5 mmol/l) to a color change to blue-violet coloration.
Calculate the concentration of chlorides in individual samples and the amount of chlorides
passed through the dialysis membrane into the beaker. Fill in the values into the table.
sample
titrationreagent standard
V
.f.Vcc
dif.Vcn
Vsample = 2.5 ml
Vdif = 150 ml, 147.5 ml, 145 ml, ... (It is getting smaller by 2.5 ml every time the sample is taken.)
Checking what has happened with the starch:
After taking of the last sample (90 minutes), stop stirring and take up the dialysis
container from the beaker. Take two test tubes and transfer a small volume (~ 1 ml) of the
solution from the dialysis container into one and from the beaker into the other one. Add 3
drops of Lugol solution (solution of iodine) into both test tubes and compare the results.
Lugol test - solution from the dialysis container:
Lugol test - solution from the beaker:
Conclusion:
33
Evaluation of the dialysis rate:
Make a graphical evaluation. Plot a graph showing increase in time of the amount of
chlorides in the solution in the beaker. First, plot the individual points according to the data in
the table. For to=0 the amount of chlorides in the beaker is no=0.Then draw optimal interlaced
curve.
axis x ... time (minutes)
axis y ... n = amount of chlorides in the beaker (mmol)
Divide the scale on axis x into 5-minute intervals (time t1, t2, t3, ...) and from the curve
read on axis y the corresponding amount of chlorides (n1, n2, n3, ...).
Time (min) 5 10 15 20 25 30 35 40 45
nx (mmol)
vx (mmol Cl-/min)
Time (min) 50 55 60 65 70 75 80 85 90
nx (mmol)
vx (mmol Cl-/min)
34
Dialysis velocity (v) is defined as the amount of Cl- passing through the semipermeable
membrane per minute.
v1 = (n1 - n0) : 5 [mmol Cl-/min]
v2 = (n2 - n1) : 5
v3 = (n3 - n2) : 5 etc.
Plot a graph of dialysis velocity in time.
axis x ... time (minutes)
axis y ... v = calculated velocity (mmol Cl-/min)
At the cross section of the curve with axis x, read the time point at which dialysis stopped due
to loss of concentration gradient (balancing of Cl- concentrations on both sides of the
membrane). By extrapolation of the curve to zero time, find out the initial velocity at the cross
section of the curve with axis y.
Time point at which dialysis stopped:
Initial velocity:
Conclusion:
35
Antioxidants
Processes happening in human body may produce reactive oxygen species (ROS) and
reactive nitrogen species (RNS), collectively called RONS (Reactive Oxygen and Nitrogen
Species). These RONS are highly reactive and comprise compounds of both radical and non-
radical nature.
Reactive oxygen species (ROS)
Radicals Non-radicals
superoxide anion (superoxide) O2•- hydrogen peroxide H2O2
hydroperoxyl radical HO2• hypochlorous acid HClO
hydroxyl radical HO• ozone O3
peroxyl radicals ROO• singlet oxygen 1O2
alkoxyl radicals RO•
Reactive nitrogen species (RNS)
Radicals Non-radicals
nitric oxide NO• nitrous acid HNO2
nitrogen dioxide NO2• dinitrogen trioxide N2O3
peroxynitrite ONOO-
alkyl peroxynitrite RONOO-
Free radicals can be defined as molecules, atoms or ions, capable of independent existence
that contain et least one unpaired electron. Free radicals may be created in a number of
ways:
1) homolytic bond cleavage
2) single-electron reduction, i.e. by adding one electron
3) single-electron oxidation, i.e. by losing one electron
Chemical reactions involving free radicals are usually chain reactions with an initiation,
a propagation and a termination step. Because of the propagation phase of the chain reaction,
free radicals have the potential to be extremely harmful to biomolecules (including DNA,
proteins, and the lipids of cell membranes). Once a free radical is generated, it can react with
stable molecules to form new free radicals from them. These new free radicals go on to
generate new free radicals, and so on.
It is not surprising that living organisms are equipped with many protection systems against
free radicals. We can distinguish:
1) enzymatic systems (e.g. superoxide dismutase, catalase, glutathione peroxidase)
2) low molecular weight antioxidants (also known as 'free radical scavengers')
A free radical scavenger is a molecule that reacts with a high energy free radical species in a
propagation step of a chain reaction, forming a more stable radical species which can be
eliminated in some way before further damage is done to biomolecules.
36
Measurement of total antioxidant capacity (TAC)
Total antioxidant capacity (TAC) describes overall abilility of the sample to resist to
oxidative stress. There are several methods for the measurement of total antioxidant capacity,
different methods may evaluate it in a different way. We will use two different methods:
A) TEAC (Trolox Equivalent Antioxidant Capacity)
This method measures antioxidant capacity of the sample, as compared to the standard
- Trolox (synthetic water-soluble analogue of vitamin E). The principle of the method is
a "scavenging" (= inactivation, decomposition) of an artificially prepared stable radical ABTS
[2,2'-azino-bis(3- 3-ethylbenzothiazoline-6-sulphonic acid)]. ABTS is converted to its radical
cation ABTS+ by addition of sodium persulfate (peroxydisulfate). This radical cation is blue
in color and absorbs light at 734 nm. Samples with antioxidant activity convert the blue
ABTS+ radical cation back to its colorless non-radical form. The reaction may be monitored
spectrophotometrically at the wavelength 734 nm.
Trolox (analogue of vitamin E)
Formation of ABTS+ radical by the action of persulfate; reaction of the ABTS+ radical with antioxidant (AOH)
B) FRAP (Ferric Reducing Antioxidant Power)
This method describes total reducing activity of the analysed sample. The principle is
a reduction of ferric complexes Fe3+-TPTZ, which are colourless, into purple ferrous Fe2+-
TPTZ products. The change in colour may be monitored spectrophotometrically at the
wavelength 593 nm. The result is given as an equivalent concentration of Fe2+ ions.
TPTZ (2,4,6-tri(2-pyridyl)-1,3,5-triazine)
37
Exercise 1: Making calibration curves
Calibration curve for TEAC method was prepared by the lab assistants, you can find it (a
plot) at your working place. You have to measure yourself just the calibration curve for
FRAP method.
Prepare calibration curv fore FRAP method (Ferric Reducing Antioxidant Power)
1) Using stock solution of iron (II) sulfate (cFeSO4 = 1 mmol/l), prepare a series of
standard solutions with different concentrations in small plastic test tubes. Calculate
the concentrations of these calibration solutions.
2) Pipette 2 ml of FRAP reagent into numbered test tubes. Concerning the standard
solutions, in all cases add 50 l in precisely same time periods (recommendation: 30
s). Exactly in 10 minutes after addition , measure the absorbance against blank (test
tube 0) at the wavelength 593 nm. (In time of addition of 50 l from test tube 0, start
the stopwatch and keep it running. After 30 s, add 50 l of standard solution 1 into the
test tube 1, and so on. Exactly at 10 minutes after starting stopwatch, set the zero
absorbance using the blank solution, at 10 minutes 30 s make the measurement of the
absorbance of standard 1, and so on.)
test tube number 0 1 2 3 4 5 6 7 8 9 10
stock sol. of FeSO4 (l) 0 50 100 150 200 250 300 350 400 450 500
distilled water (l) 500 450 400 350 300 250 200 150 100 50 0
cFeSO4 (mmol/l) 0
A593 0
3) Plot calibration graph A593 = function of cFeSO4 axis y … A593, axis x … cFeSO4
38
Exercise 2: Measurement of total antioxidant capacity of different samples
You will measure total antioxidant capacity of three different samples by two methods
(TEAC and FRAP). First, you have to prepare the samples:
sample 1 – blood serum – it is ready to use at your working place
sample 2 – saliva
Do not eat or drink during the 30 minutes preceding collection. Open the Salivette tube
and put the cotton roll into your mouth. Gently chew the cotton roll and swirl it around the
mouth for about 1 minute until the cotton is saturated in saliva. Place the saturated cotton
roll back inside Salivette tube and close the lid. Centrifuge at 2.500 RPM for 2 minutes.
sample 3 – commercially available antioxidant supplement ("miraculous pill")
In a mortar, crush a pill of antioxidant supplement. Transfer a powder to a small beaker
and dissolve it in 5 ml of distilled water. Prepare filtration apparatus (funnel with filtrate
paper) and and use it to remove insoluble deposits. Transfer 0.5 ml of the filtrate in a
beaker and dilute it with 100 ml of distilled water. This diluted solution of antioxidant
supplement is the sample 3.
A) TEAC method
Pipette 2 ml of ABTS reagent into numbered test tubes. Concerning the samples, in all cases
add 50 l in precisely same time periods (recommendation: 1 minute). Exactly in 10 minutes
after addition of the sample, measure the absorbance against blank (test tube 0) at the
wavelength 734 nm. Using the calibration curve for TEAC method, read the corresponding
values of trolox concentration.
test tube number - sample 0 1 - serum 2 - saliva 3 - pill
ABTS reagent (l) 2000 2000 2000 2000
sample (l) - 50 50 50
distilled water (l) 50 - -
A734 0
cTROLOX (mmol/l) 0
B) FRAP method
Pipette 2 ml of FRAP reagent into numbered test tubes. Concerning the samples, in all cases
add 50 l in precisely same time periods (recommendation: 1 minute). Exactly in 10 minutes
after addition of the sample, measure the absorbance against blank (test tube 0) at the
wavelength 593 nm. Using the calibration curve for FRAP method, read the corresponding
values of FeSO4 concentration.
test tube number - sample 0 1 - serum 2 - saliva 3 - pill
FRAP reagent (l) 2000 2000 2000 2000
sample (l) - 50 50 50
distilled water (l) 50 - -
A593 0
cFeSO4 (mmol/l) 0
39
Exercise 3: Measurement of total antioxidant capacity of vitamin C solutions
Using stock solution of vitamin C (c=1 mmol/l), prepare a series of solutions with different
concentrations in small plastic test tubes:
test tube number 0 1 2 3 4 5
stock sol. of vit. C (l) 0 100 200 300 400 500
distilled water (l) 1000 900 800 700 600 500
cvitamin C (mmol/l) 0
A) TEAC method
Pipette 2 ml of ABTS reagent into numbered test tubes. Concerning the samples of vitamin C,
in all cases add 50 l in precisely same time periods (recommendation: 1 minute). Exactly in
10 minutes after addition of the sample, measure the absorbance against blank (test tube 0) at
the wavelength 734 nm. Using the calibration curve for TEAC method, read the
corresponding values of trolox concentration.
test tube number 0 1 2 3 4 5
ABTS reagent (l) 2000 2000 2000 2000 2000 2000
vit. C sample (l) 50 50 50 50 50 50
A734 0
cTROLOX (mmol/l) 0
B) FRAP method
Pipette 2 ml of FRAP reagent into numbered test tubes. Concerning the samples of vitamin C,
in all cases add 50 l in precisely same time periods (recommendation: 1 minute). Exactly in
10 minutes after addition of the sample, measure the absorbance against blank (test tube 0) at
the wavelength 593 nm. Using the calibration curve for FRAP method, read the
corresponding values of FeSO4 concentration.
test tube number 0 1 2 3 4 5
FRAP reagent (l) 2000 2000 2000 2000 2000 2000
vit. C sample (l) 50 50 50 50 50 50
A593 0
cFeSO4 (mmol/l) 0
40
Enzymology I
1) Enzyme specificity
Introduction:
Most of the enzymes are highly specific for the reaction to be catalyzed and for the
substrate to be selected. This property will be demonstrated in reaction with adequate
substrate. We shall demonstrate also the thermolability of enzymes. We shall use α-amylase
from saliva and sucrase of baker´s yeast (Saccharomyces cerevisiae). Sucrase hydrolyses β-
glycosidic bond of sucrose to glucose and fructose. This enzyme is different from sucrose in
the intestinal juice of mammals which hydrolyses α-1 glycosidic bond.
Procedure:
1. Take approximately 0.5 g of yeast and suspend in 2 ml of distilled water. This is the
sucrase solution.
2. Take diluted saliva from previous experiment as amylase solution.
3. Prepare a set of 6 test tubes and pipette according to the table. Perform the
described experiment:
Test tube number 1 2 3 4 5 6
Amylase (ml) 0.5 0.5 0.5
Sucrase (ml) 0.5 0.5 0.5
Boil (ml) yes yes
Starch (ml) 2.0 2.0 2.0
Sucrose (ml) 2.0 2.0 2.0
Place into the water bath (37°C) for 30 minutes. Then divide the content of each tube into two parts. The first portion will be examined with Fehling reagent (mix 2 ml of Fehling I + 2 ml of Fehling II, add 0.5 ml of this solution to each tube and boil it) and second with iodine (1 drop of iodine solution + 5 ml of water). Recorde the result:
With Fehling r.
With iodine
41
4. Evaluate the result in each test tube and make conclusions in your protocol:
Tube No
Explanation
1
2
3
4
5
6
42
2) Dependence of activity of α-amylase on pH
Introduction:
Most of the enzymes are strongly pH-dependent in their activity. This will be
demonstrated on α-amylase which is present in the saliva of oral cavity and in the pancreatic
juice. It hydrolyses starch and glycogen to maltose, maltotriose, and a mixture of branched
oligosaccharides, and non-branched oligosaccharides. At pH 4 the enzyme is completely
inactivated.
Procedure:
1. Prepare a set of 7 test tubes and mark them. Pipette the buffers according to the
table:
Test tube Na2HPO4 (ml) Citric acid (ml) pH Colour
1 2.9 2.1 5.6
2 3.1 1.9 6.0
3 3.5 1.5 6.4
4 3.8 1.2 6.8
5 4.3 0.7 7.2
6 4.7 0.3 7.6
7 4.9 0.1 7.8
2. Add 1 ml of 1% starch solution (not containing buffer!) into each tube as substrate.
3. About 0.5 ml of saliva dilute with 6 ml of water (not buffer). Add 0.5 ml of diluted
enzyme solution to each tube and leave in water bath (37°C) for 10 minutes.
4. After 10 minutes of incubation pour approximately half of the reaction mixture into
another set of tubes and the remaining half leave on the water bath for another 5
minutes.
5. To each of the samples taken of the incubation add 5 ml of water and 1 drop of
iodine solution (Lugol). Register the colour that develops.
6. After the second incubation is completed take the samples of the bath, add again 5
ml of water to each tube and again 1 drop of iodine. Register the colour again.
7. Evaluate the results:
What pH is the most suitable for α-amylase?
43
3) Estimation of alpha-amylase in urine
a) by the test – BIOSYSTEMS – EPS
Principle of the method:
Amylase catalyzes the hydrolysis of synthetic substrate (4- nitrophenyl-
maltoheptaoside-ethylidene) to smaller oligosacharides which are hydrolyzed by α-amylase
liberating 4- nitrophenol. The amount of arising 4-nitrophenol correspond with the catalytic
concentration of alpha-amylase.
Procedure:
1. Switch on photometer ECOM E 6125 and the printer. Deprres the key ABSORBANCE
and with arrow keys set the wavelength to 405nm. Rinse several times the cuvette
in phofometr with distilled water. The cuvette is emptied with the pump.
(Ask the lab assistant for the demonstration how to use it.)
2. Stop the pump and fill the cuvette with distilled water as blank. Check the display of
the photometr. It should show ABSORBANCE 405 nm BLANK. If not, used the key
BLANK to obtain this parameter. Depress the key ENTER- MEASURE, the absorbance
on the display should be 0,000.
3. Sample measurement: Mix into Eppendorf tube - 20µl of urine and 1000µl of the
“Reagent solution “, immediately transfer into cuvette and start the stop-watch.
Measure the absorbance of sample exactly after 2nd, 3rd, 4th and 5th minute. The
measurement is achieved by depressing the key ENTER-MEASURE. The result of
measurement is always printed out.
4. Rinse several times the cuvette with distilled water and switch of the pump,
photometer and printer.
5. From the values obtained, calculate the average ΔA/min. and substituting in formula
concentration α-amylase in the urine sample.
Calculation: Urine measurements at 37⁰C (reaction temperature) ∆A1= A3 – A2 ∆A2= A4 – A3 ∆A3= A5 – A4 mathematical average ∆A /min = ∆A1+ ∆A2+ ∆A3 / 3
µkat/l = ∆A /min x 105,9 (conversion factor)
U/L = ∆A /min x 6384 (conversion factor)
Benchmarks at 37⁰C µkat/l = 0,26-8,15 U/L = 16 - 491
44
b) Wohlgemuth´s test
Procedure:
1. Into a series of ten test tubes pipette 1 ml of 0.9% NaCl with the exception of the
first test tube.
2. Into tube No 1 and 2 place 1 ml of urine.
3. Mix the content of tube 2 and take over 1 ml to the third test tube. Mix, measure
off again 1 ml and in this way continue the dilution of urine in the following test
tube. The last 1 ml volume from tube No 10 should be discarded.
45
4. To each test tube add 2 ml of the buffered 0.1% starch solution. Allow incubate in
a water bath at 45°C for 15 min.
5. Cool test tubes and add into each tube 3 drops of iodine solution. Examine the
coloration of the content. Record the number of the last test tube which gives no
blue colour with iodine.
6. Calculate the activity in Wohlgemuth units corresponding the volume (in ml) of
0.1% starch digested by 1 ml of urine under conditions described.
For example: if ¼ ml of urine (dilution 4x) digested 2.0 ml of starch, the enzymic activity equals 2 x 4 = 8 U. The Wohlgemuth units correspond very roughly to µkalt/l. Complete urine dilution:
1x 2x
Conclusion:
46
Enzymology II
1) Estimation of Michaelis' constant 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 [l] 450 400 350 300 250 200 150 100 50
Substrate [l] 50 100 150 200 250 300 350 400 450 500
3. Place the set of test tubes into the water bath at 37C for 5 minut. Meanwhile, bring
the enzyme from the refrigerator, prepare stop watch and the pipette of 50 l with a
clean tip.
4. Enzyme solution must be pipetted in absolutely exact 20 seconds period. Add 50 l 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
47
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.
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.
48
2) 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).
49
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.
50
Enzymology III
1) 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
51
2) Alanine aminotransferase and aspartate aminotransferase 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.
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.
52
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. Evaluate the results.
53
3) Estimation of alkaline phosphatase in blood serum
Principle:
Alkaline phosphatase (ALP) 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.
54
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 (kat/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)
55
Enzymology IV
1) 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), K3[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.
56
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 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.
57
2) 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
sample
Test tube No. 2
blank
Test tube No. 3
sample
Test tube No. 4
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.
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.
58
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
59
3) 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.