UNIVERSITY OF LILLE – LICENCE SVTE (L1 S2)
Biochemistry 1 : Molecules of Life - Practical
It is mandatory to wear safety glasses during all experiments. Glasses and propipetters borrowed at
the beginning of the practical must be returned at the end of the session.
Practical 1 – Amino acids and proteins
I – PRESENTATION, INSTRUCTIONS AND DEMONSTRATION
• Presentation of glassware; laboratory rules; security instructions
• Use of equipment: propipetter, automatic pipette, scales, spectrophotometer, dispenser, vortex,
magnetic stirrer, centrifuge
II – THIN LAYER CHROMATOGRAPHY OF AMINO ACIDS
Principle
Thin layer chromatography (TLC) is a very sensitive analytical method very commonly used in
biochemistry. Separation is performed on a solid, inert and hydrophilic support (thin layer of silica gel)
via the ascension of a solvent mixture by capillarity. Amino acids have different partition coefficients
between the support (to which they can bind reversibly, which is known as adsorption) on the one hand
and water and other solvents on the other hand. This allows them to be separated.
Visualization of amino acids after migration is performed using ninhydrin, leading to the formation of a
purple chromophore (Ruhemann's purple) for all amino acids except proline, for which a yellow
chromophore is formed.
Experimental
- On a TLC plate (4 × 10 cm) covered with silica gel (0.2 mm thickness), using a pencil, carefully draw
a horizontal line at 1.5 cm distance from the bottom. Samples will be deposited on this line at a
spacing of about 1 cm.
- 3 samples: Each team deposits 1 µl of sample A or B containing a mix of 3 amino acids at a
concentration of 2.5 mg/ml. (Sample A: Arg/Pro/Trp ; sample B: Val/Asp/Lys). The two other deposits
correspond to 2 control amino acids chosen among the following six: Trp, Val, Asp, Arg, Lys, Pro.
- Dry the spots (hairdryer) and place the TLC plate in the separation chamber containing the migration
solvent (n-butanol/acetic acid/water, 75/20/20). Close the chamber and let migrate for about 2 hours
until the solvent reaches the top of the TLC plate.
- Remove the TLC plate and locate the migration front.
- Dry the plate and spray on the ninhydrin solution (1% ninhydrin in acetone) under the fume hood.
- Heat the plate until staining appears.
Results and interpretation
Circle the dots that you observe, note their color. Identify the amino acids present in your sample
and justify your answer. Calculate their Rf values.
III – PROTEIN QUANTIFICATION – SPECTROPHOTOMETRY
Principle
The principle of quantification of a substance by spectrophotometry is applied to the colorimetric
quantification of proteins using the biuret test. After determination of the wavelength at which the
absorption of a compound is maximal, its absorbance (A) can, under certain conditions, be related to its
concentration (c in mol/L) using the Beer-Lambert law: A = ε · l · c (with l the optical path length in cm
and ε the molar extinction coefficient of the compound at the given wavelength in
cm-1 · L · mol-1).
The colorimetric quantification of proteins via the biuret test is based on the biuret (Gornall's) reagent,
which reacts with peptide bonds. Under alkaline conditions and in presence of copper(II), a purple color
appears. Its intensity can be measured via absorbance at 540 nm by spectrophotometry. This intensity is
related to the concentration of the colored compound formed and, therefore, to the initial protein
concentration.
Experimental
A serum albumin solution at 10 g/L is used to prepare a series of solutions with varying known protein
concentrations (standard range – to yield a calibration curve). After reaction with biuret reagent, the
absorbance of these solutions as well as the absorbance of the solution to quantify (solution X) is
measured. Glass tubes are used for the measurements. The following table indicates volumes in mL.
Standard range Solution X
Tube number 1 2 3 4 5 6 7 8
Serumalbumin at 10g/L 0 0.2 0.4 0.6 0.8 1 - -
NaCl at 8g/L 1 0.8 0.6 0.4 0.2 0 0.5 0
Solution to quantify - - - - - - 0.5 1
Biuret reagent 4 4 4 4 4 4 4 4
Homogenize thoroughly after addition of each solution (vortex) and incubate for 30 minutes in the dark.
Read the absorbance at 540 nm using plastic cuvettes. Set the absorbance to zero using the blank (tube 1).
The biuret reagent is available ready-to-use in a dispenser. It has been prepared as follows:
1.5 g CuSO4 were dissolved in 200 ml water. 6 g sodium potassium tartrate and 30 g NaOH were added.
After dissolution and mixing, 1 g potassium iodide was added, and the volume was completed to 1 l with
water. The reagent is stored in the dark in a tight flask.
Results and interpretation
Graph the curve A = f(amount of protein per tube in mg).
Using the graph, calculate the protein concentration in g/L of solution X.
IV – PRECIPITATION OF PROTEINS
Principle
Denaturing precipitation of proteins can be obtained using acids (HCl or TCA = trichloroacetic acid) or
organic solvents (ethanol, acetone).
Non-denaturing precipitation of proteins is performed by "salting out" using neutral salts such as
ammonium sulfate. Proteins may be resolubilized after elimination of the precipitating agent.
Experimental
- Mix 1.5 ml 1% ovalbumin solution and 1.5 ml 20% HCl in a plastic tube (cover with parafilm for
mixing).
- In a second plastic tube, mix 1.5 ml 1% ovalbumin solution and 1.5 ml saturated ammonium sulfate
solution ((NH4)2SO4 at about 750 g/l), using parafilm to cover the tube.
- Place the tubes at 4°C for at least 30 minutes to favor precipitation.
- Centrifuge for 10 minutes at 3000 revolutions per minute (rpm)
- For each tube, pour away the supernatant and redissolve the pellet in 3 ml distilled water.
Results and interpretation
Compare the results of the two precipitations.
At the end of the practical, hand in the report form (one for each team) completed with calculations
and interpretations, as well as the protein quantification graph and the annotated TLC plate.
Do not forget to indicate your names and your team number as well as the identification numbers
of the solutions you analyzed (amino acid mixture; solution X used in quantification).
Practical 2: Carbohydrates
I – MEASUREMENT OF GLUCOSE CONCENTRATION BY THE GLUCOSE OXIDASE
ASSAY
Principle:
The concentration of glucose in a solution is measured using the glucose oxidase assay (a method
commonly used to determine blood sugar levels), using a calibration solution at 1 g/l. The reaction
scheme for the assay is as follows:
(1) Glucose + O2 → Gluconic acid + H2O2
(2) 2 H2O2 + phenol + 4-amino-antipyrine → quinone imine + 4 H2O
Reaction (1) is catalyzed by glucose-oxidase, reaction (2) by peroxidase. The intensity of the resultant
coloration is measured on a spectrophotometer by absorbance at 505 nm.
Experiment:
The calibration solution of glucose at 1 g/l is prepared by the instructors as a demonstration for all
groups (weighing in of glucose; preparation in volumetric flask).
The reagent for glucose determination is provided in a dispenser. Its composition is as follows:
Phosphate buffer pH 6.6...................... 225 mM
Amino-4-antipyrine .............................. 0.3 mM
Phenol................................................... 8.5 mM
EDTA ...................................................... 5 mM
Peroxidase ...................................... > 300 U.L-1
Glucose oxidase ........................ > 10 000 U.L-1
Each group has its own glucose solution whose concentration is to be determined (sample X). Perform
the assay in plastic hemolysis tubes according to the following table (for µL volumes, use an automatic
pipette):
Tube 1 : Zero / control Tube 2 : Calibration Tube 3 : Measurement
Distilled water 10 µL - -
Calibration solution - 10 µL -
Sample X - - 10 µL
Reagent 1 mL 1 mL 1 mL
Mix, incubate 20 min at room temperature (the dye is stable for about 1h).
Read absorbance at 505 nm against the zero solution using a microcuvette.
Results:
By comparing absorbances, determine the glucose concentration of sample X.
II – STARCH HYDROLYSIS
Principle:
Starch is a homopolysaccharide composed of glucose molecules linked in α-1,4 and α-1,6 (for branching
points).
Starch is insoluble in cold water, but forms a suspension called starch paste in hot water. In the presence
of iodine solution, starch gives a blue color.
α-amylase is an enzyme that hydrolyzes starch to dextrins. The coloring obtained by fixation of iodine on
dextrins depends of the length of the glucose chains. This way, using a color scale of iodine staining (blue
– red – yellow), one can follow the evolution of the enzymatic hydrolysis of starch.
Preparation of the starch paste:
This protocol allows for the preparation of an amount of starch paste sufficient for 4 groups. The
resultant suspension is distributed into 4 beakers before being stored at 4°C.
Weigh 8 g of starch in a dish and add 60 ml of deionized water. Stir and pour this suspension into a
beaker containing 100 mL of deionized water that has previously been boiled. Keep boiling gently and
mix carefully to generate a homogeneous suspension. Let cool down, distribute into 4 beakers and keep at
4°C for at least 15 min before using for hydrolysis. Do not forget to mark each beaker with your group
number.
Enzymatic hydrolysis:
Prepare 15 test tubes, each containing 15 ml of deionized water (measured in a graduated cylinder) with 5
drops of iodine-iodide solution added (ready-to-use solution: potassium iodide 20 g, iodine 10 g, add
water to a final volume of 1 l). Mix to obtain a homogeneous pale yellow color.
Start the enzymatic digestion by adding 0.1 to 0.2 mL of α-amylase solution (to be kept on ice!) to the
refrigerated starch suspension. Mix with a glass stirrer.
To take the first sample, dip the tip of the stirrer briefly into the first test tube with iodine solution and
mix briefly back and forth, then withdraw. Shake the tube and clean the stirrer. Continue stirring the
starch suspension, which starts to liquefy due to the action of the enzyme. At regular time intervals (first 1
min, then 30 s depending on how the colors in the tubes develop), repeat the sampling procedure with the
stirrer and the next test tube. Follow the evolution of the color scale until it returns to its initial yellow
color.
III – MEASURING PIGMENTS CONTAINED IN SYRUPS
Experiment:
The absorbance of pigments present in a syrup (mint or grenadine) is measured on a spectrophotometer as
a function of the dilution of the syrup.
Each group performs 3 dilutions (twice for each dilution) with one of the two syrups according to the
following protocol: sample the syrup using an automatic pipette, prepare 2 ml of each dilution, read the
absorbance in a plastic cuvette on the spectrophotometer.
- Mint syrup (brilliant blue): 1/5, 1/10, and 1/20 dilutions, absorbance measured at 630 nm.
- Grenadine syrup (anthocyanes): 1/2, 1/4, and 1/5 dilutions, absorbance measured at 525 nm.
Plot the graph: absorbance = f (dilution) for the syrup used.
At the end of the practical, present the color scale you obtained in the starch hydrolysis.
Hand in a results sheet (one per group, with your names and your group number) comprising the
glucose quantification (mentioning the number of your sample X) and the absorbance values
obtained for the syrup dilutions, along with the corresponding graph.
