Faculty of Sciences Biochemistry and Molecular Biology

2

1. Assistance to the Laboratory is mandatory. The lack of any Lab session not justified

by writing will suppose not being able to hand in the final report and, therefore, not

getting the grades part of the on-going assessment.

RECOMMENDATIONS

2. It is compulsory to use safety glasses in the laboratory, so each person must have

their own.

3. The students must wear a laboratory coat all the time (each person must have their

own lab coat) and preferably safety glasses when doing practical work.

4. Must not be work with flammable products near lit Bunsen burners.

5. All eating and drinking in the laboratory is, of course, strictly forbidden.

6. Be careful with products and toxic solvents. Never pipette poisons by mouth, use a

safety bulb (pro-pipette).

7 It is necessary to take to the laboratory, besides the corresponding scripts of the

practical exercises, a notebook to note down the calculations, results and the

observations that you are obtaining.

8. Prior to throwing away any product down the sink open the water taps. On the

other hand, there are many products that should not be put down the sink (such as

ethanol, azide, sulphuric acid, phenol ...); in these cases we provide you with bottles

where they can be stored for later disposal. PLEASE, ASK YOUR SUPERVISOR BEFORE

YOU THROW AWAY ANY PRODUCT.

9. Before carrying out a practical exercise you should review all materials and reagents

needed for it, indicating any abnormality.

10. Clean the material before use, and then rinse with distilled water. After the lab

session, you have to leave everything perfectly clean and ordered. At the end of each

practical session we will review the state of the material.

11. As a security measure, control the Bunsen burner keys before opening the gas tap.

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12. If you need to heat the contents of a test tube directly over the Bunsen burner the

test tube will be somewhat inclined with stirring and with the tube mouth directed

toward a place where there is no person.

13. The common material, as well as general reagent bottles should be placed in their

place immediately after use.

14. To avoid contamination do not pipette reagents directly from bottles of common

usage. Put a small amount into a glass and pipette from there.

15 The reagents which have been taken out of bottle and have not been used must not

be poured back into the bottle, since the content can be contaminated. Therefore, the

amounts of reagents are removed from the bottles should not exceed those necessary

for the practical exercises.

16. When you have to dilute acid using distilled water, always add the acid first.

17. Reactions with any harmful gas production are primarily carried out in the

chemical fume hood with the suction system in operation. The atmosphere of the

laboratory must be kept as clean as possible.

Criteria to evaluate Lab reports

Format 10% Principles 10%

Results 50% Conclusions 20% References 10%

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HAZARD SYMBOLS

FLAMMABLE SUBSTANCES Self-heating substances and mixtures. Substances and mixtures, which in contact with water, emit flammable gases. TOXIC SUBSTANCES (Skull and Crossbones) Acute toxicity (oral, dermal, inhalation). Products, which inhaled, ingested or in contact with skin, cause injury or death.

CORROSIVE SUBSTANCES. Contacts with these products destroy living tissues and other materials.

IRRITATING OR HARMFUL SUBSTANCES. Irritants: They act on the skin, eyes and respiratory tract. Harmful

: The absorption of these products has led to minor injuries

OXIDISING SUBSTANCES Products which in contact with others, particularly with flammable substances, originate a highly exothermic reaction. EXPLOSIVE SUBSTANCES Products that are more sensitive to shocks than dinitrobenzene or they may explode under the effect of flame or friction.

5

GEL FILTRATION CHROMATOGRAPHY. SEPARATION OF HAEMOGLOBIN AND

ALANINE IN SEPHADEX G-25.

Gel filtration chromatography is a method for separating proteins and peptides based

on their size. The chromatographic matrix consists of porous beads, and the size of the

bead pores defines the size of macro-molecules that may be fractionated. Those

proteins or peptides that are too large to enter the bead pores are “excluded,” and

thus elute from the column first. Since large molecules do not enter the beads, they

have less volume to pass through. Because of that reason, they are the first to elute

from the column. Smaller macromolecules that enter some, but not all of the pores are

retained slightly longer in the matrix and emerge from the column next. Finally, small

molecules filter through most of the pores, and they elute from the column with an

even larger elution volume. This method is also called gel permeation, molecular sieve,

gel-exclusion, and size-exclusion chromatography. Since no binding is required and

harsh elution conditions can be avoided, gel-filtration chromatography rarely

inactivates enzymes, and often is used as an important step in peptide or protein

purification.

1. PRINCIPLES

Gel filtration is a simple and reliable chromatographic method for separating

molecules according to size. Its versatility makes it generally applicable to the

purification of all classes of biological substances, including macromolecules not

readily fractionated by other techniques.

This technique involves the separation of molecules with different sizes through

a gel column. The polysaccharide dextran is cross-linked giving small portions of a

hydrophilic and insoluble polymer that swells in water to form a gel. Small molecules

can penetrate into the gel, but large are excluded from cross-linked network. First, the

mixture of large and small molecules is placed on the top of the column (of the gel). As

they pass down the column, the small molecules diffuse into and following a longer

path than the large molecules, which are completely exclude from the gel particles.

Eventually, complete separation occurs, with the larger molecules leaving the column

first and the smaller ones last.

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We can distinguish some constants which are function of the type and size of

the gel bead. These constants are:

Ve = the elution volume of a compound is the volume required to elute that

compound from a column. The elution volume of the molecule, in mL, is the volume of

the eluted solvent necessary to get the higher concentration of the molecules of

interest.

Vo = void volume. It is the elution volume of a molecule which is completely

excluded from the gel, in other words, V0 is the volume of mobile phase between the

beads of the stationary phase inside the column.

Vt = Total volume (Vt) is calculated from the volume of the column bed (πr2 x

length). To calculate it, we need to know the height and the section of the column.

Kav = Ve - Vo/Vt - Vo

Kav = is the partition coefficient between the liquid phase and the gel phase.

This variable is independent of geometry of the column the compacting of gel bed and

exists an almost linear relationship between Kav values for various substances and the

respective logarithms of their molecular weights, by which, knowing Kav you can have

an approximate knowledge of the molecular weight. Kav values are plotted against the

log of the molecular weight for each protein standard.

The pore size is typically determined to the extent of cross-linking between the

polymers of the gel material which determines the fractionation range of molecular

sizes. This resin allows separate globular proteins whose molecular weight is between

1000 and 5000 Da. The latter means that molecules larger than 5000 Da will be unable

to enter the pore of the matrix.

In this experiment we observe the separation of two molecules based on the

size differences. Initially, a mixed of blue dextran and potassium chromate is passed

trough. The blue dextran has a molecular weight of 2 • 106 Da, larger than the range of

Sephadex fractionation, which is not retained and eluted first being unable to enter

7

the pores of the matrix. Potassium chromate has a molecular weight of 194 Da, less

than the Sephadex fractionation range, thus will be retained and eluted as eluent pass

down the column.

Then add a mixture of haemoglobin (MW 66000 Da), and alanine (MW 89 Da)

to the top of the column. As the limit fractionation of the gel is between 1000 and

5000 for peptides and proteins, haemoglobin will not be retained and eluted with the

void volume of the column. In contrast, alanine will be retained, eluting practically at

the same volume as the chromate.

Due to the fact amino acid is colourless its presence is confirmed by the

ninhydrin test. Ninhydrin is a powerful oxidising agent that reacts with α-amino acids

at a pH between 4 and 8, resulting in the formation of ammonia and CO2, reducing

ninhydrin to hydridantine. This compound reacts in turn with ammonia and ninhydrin

to give a double addition compound deep purple-blue. This reaction is very sensitive.

2. MATERIAL.

* Chromatography column Sephadex G-25

* 250 ml Erlenmeyer

* Pasteur pipettes

* 0.5 mL pipette (2 units)

* 25 mL Graduated Volumetric Cylinder

* Test tubes and a rack

* Colorimeter or spectrophotometer

* Water Bath

3. SOLUTIONS

* Haemoglobin (2 mg/mL)

* Alanine (6 mg/mL)

* Ninhydrin (5 mg/mL)

* Potassium chromate (2 mg/mL)

* Blue Dextran (10 mg/mL)

* Sodium chloride, NaCl (10 g/L)

8

4. METHOD

Leave the solution of NaCl, which is inside the column at the edge of the gel, and now

add a mixture (previously prepared in a test tube) of 0.5 mL of potassium chromate

and 0.5 mL of blue dextran to the top of the column. Allow the mixture to penetrate

the column and collect 3 mL fractions. At the time this solution has reached a level of

gel, NaCl solution is added to the Pasteur pipette and then you can connect the

capillary to the flask containing NaCl. The flow rate should be slow. The

chromatography is finished when all the chromate has been eluted. Then we measure

the absorbance of these tubes, the first test tubes (blue) at 621 nm, and the following

(yellow) at 470 nm. Tube 1 or NaCl solution is used as blank.

NOTE: THE COLUMNS MUST ALWAYS HAVE LIQUID ON THE TOP OF THE GEL

Then a mixture with 0.5 mL of haemoglobin solution and 0.5 mL of alanine

solution is prepared in a test tube. Again the liquid level is carried out up to the level of

the gel and then this mixture is added with a Pasteur pipette and operating the same

way as before. The chromatography may have ended when many tubes are taken as in

the previous step.

The absorbance of these tubes is measured at 430 nm (against tube nº1 or NaCl

solution as the blank) up to the value returns to be 0 (it is left to measure), these tubes

are those containing haemoglobin. All tubes that follow are made to the ninhydrin

test, as contained alanine.

To do this, add 10 drops of this reagent to each tube, shake well and incubate

in a boiling water bath until a blue colour appear in some test tubes. After this time,

allow to cool and where the amino acid will be bluish discoloration appear. Then, pass

all measuring absorbance at 540 nm.

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5. CALCULATIONS.

To measure the volumes is done as follows:

a) It joins as the sample is placed until the first coloured drop elutes from the

column (A).

b) It joins the entire volume of the coloured substance (B).

To calculate the elution volume, it should be assumed that the elution process

is symmetrical, so that we can imagine that the maximum concentration of the

substance in the elution will be in the middle of the volume containing the coloured

substance.

With this assumption, the elution volume for each substance will be:

Ve = A + 1/2 B

6. QUESTIONS

1. Do chromatograms of the results (plotting test tube number (or volume) on

x-axis and absorbance on y-axis).

2. Measuring the elution volumes of each substance and calculate the different

values of Kav (diameter of the column = 1.5 cm. You have to measure the height of gel

in the column).

3. Depending on the molecular mass, what proteins eluted in last place from

the column, the larger or smaller?

4. Define briefly the following concepts:

a. Partition coefficient. b. Void volume.

c. Elution volume. d. Total volume.

5. What is the void volume of this column?

6. Explain what the mobile phase and stationary phase in chromatography are .

7. Why is ninhydrin employed in the practice of the gel filtration?

8. What is the function of the blue dextran in the gel filtration practice?

9. What is the function of the potassium chromate in the practice of gel

filtration?

10. What is the relationship between the molecular weights of the various

substances and their partition coefficients?

10

ISOLATION OF CASEIN AND LACTOSE FROM MILK

1. PRINCIPLE.

Casein is the main protein found in milk and is present at a concentration of

about 35 g/L. It is actually a heterogeneous mixture of phosphorous containing

proteins and not a single compound. It is a phosphoprotein existing in four forms:

alpha, beta, gamma and kappa casein, each one with different composition.

Like many other proteins derived from animal sources, e.g. meat (myosin) and

eggs (albumin), casein is a nutritionally adequate protein. These proteins contain all

essential amino acids required for normal growth and development.

Most proteins show a minimum solubility at their isoelectric point and this

principle is used to isolate the casein by adjusting the pH of the milk to 4.8, its

isoelectric point. Casein is also insoluble in ethanol and this property is used to remove

unwanted fat from the preparation.

2. EQUIPMENT.

* 2 beakers (250 mL)

* 3 Graduated volumetric cylinder

100 mL, 50 mL and 25 mL

* Glass rod

* Thermostatic bath at 40º C

* pHmeter or pH paper

* funnel and tripod

* Pipette (2 mL)

* Buchner filter equipment and

papers

* Büchner flask

* Foil

* Muslin

3. MATERIALS.

* Fat-free milk

* Glacial acetic acid

* Ethanol (96% v/v)

* Charcoal

* Calcium carbonate

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4. PREPARATION OF SOLUTIONS.

* 10% Acetic acid: 18 mL distilled water and 2 mL glacial acetic acid.

5. METHOD.

5.1. ISOLATION OF CASEIN:

Place 100 mL of milk in a 250 mL beaker and warm to 40ºC. Add slowly 10%

acetic acid, without shaking, and when it contains a few millilitres of acid is stirred

with the rod for a perfect mix of acid and milk. The final pH of the mixture should be

around 4.8 and this can be checked with a pH meter or pH paper. A white precipitate

of casein is produced. Cool the suspension to room temperature and leave to stand for

a further 5 minutes before filtering through muslin (three layers).

Wash the precipitate several times with a small volume of distilled water then

suspend it in about 30 mL of 96% ethanol. Filter the suspension on a Buchner funnel

and wash the precipitate a second time with 25 mL of ethanol and suck dry.

Remove the white powder and spread out on a piece of foil to allow the

ethanol to evaporate (if possible, leave a few minutes in an oven).

5.2. ISOLATION OF LACTOSE:

Add 3 g of powdered calcium carbonate. Stir the mixture thoroughly to obtain a

good suspension, heat the mixture in a boiling water-bath for about 10 -15 minutes;

stirring continuously. This should precipitate the remaining proteins of milk.

Filter the hot mixture, collecting the filtrate in a 250 mL glass beaker and stir

the filtrate continuously while boiling it down to about 10mL. After that, add 50mL of

95% Ethanol. After filtering the hot mixture filter paper with funnel, and discard the

precipitate, the filtrate is passed to another 250 mL glass beaker and subjected to

boiling on a Bunsen burner (it is convenient to introduce a few ‘glass beads’ to get a

homogeneous boiling). In case there is a foam formation, it can be eliminated by

shaking strongly with a clean glass rod. Boiling is continued until to have 20 to 25 mL of

liquid.

When the volume of the sample is reduced up to 20-25 mL, the glass beaker

should be removed from the flame. Then, add 100 mL of 96% ethanol to the syrup still

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warm and approximately 1 g of active carbon powder (to discolour the dark syrup). Stir

well to get a homogeneous mixture. It is filtered through double filter paper and the

clear filtrate is collected (it is critical that the liquid is still warm to prevent premature

crystallisation of the lactose). The clear filtrate is cooled in the freezer for several days

(at least 2 days) to allow the lactose to crystallise . Granular crystals will form during

this time. The lactose crystals can be separated by filtration. After drying by air or in

the oven at about 60 ° C, the lactose obtained has to be weighed to calculate the yield.

It must be taken into account that the lactose obtained is lactose monohydrate (Pm =

360.42 Da), so the water content in the molecule must be used at the time of

calculating the purity of product obtained.

6. QUESTIONS.

1. Weigh the casein and calculate the percentage yield of the protein

2. Why do you use fat-free milk?

3. Weigh the lactose and calculate the yield.

4. Why should you add the charcoal?

5. When you separate casein from lactose, why is calcium carbonate added to

lactose powder?

6. What is the effect of ethanol on lactose once you obtain the lactose syrup?

7. What principle of the proteins is used to isolate the casein?

13

ENZYMATIC ACTIVITY. CATALASE

1. PRINCIPLES.

In higher organisms, catalase occurs in cell organelles called peroxisomes. It is

one of the fastest reacting enzymes known. It catalyses the breakdown of hydrogen

peroxide to water and oxygen. Superoxide radicals (O2-) are generated during aerobic

respiration when a small amount of the oxygen that normally forms water gains an

electron. Excess superoxide may be converted to more damaging hydroxyl radicals.

Within cells the enzyme superoxide dismutase removes superoxide by converting it to

hydrogen peroxide. Hydrogen peroxide is toxic to the body and is broken down by

catalase. Hydrogen peroxide is also produced by white blood cells. They produce it

during phagocytosis to kill microorganisms.

Catalase is an enzyme found in all living cells (with some exceptions, among

anaerobic microorganisms), but is especially abundant in blood and liver. It has been

prepared in crystalline form from both sources. The enzyme is highly specific and acts

only on the H2O2, which is the substrate, although alkyl peroxides used as electron

donors. Catalase catalyses the following reaction:

H2O2 H2O + ½ O2

Hydrogen peroxide is a product of different cellular oxidations, and catalase is

present in cells to prevent accumulation of H2O2. It is one of the most active known

enzymes.

The activity of catalase has been determined by different methods. One

convenient, although approximate, is to determine the undecomposed H2O2 by

titration with permanganate after incubating the enzyme with excess H2O2. Is allowed

to act the enzyme in a dilute solution of peroxide during 5 minutes and the reaction is

stopped by the addition of sulphuric acid that destroys the enzyme. The titration

reaction is:

2 MnO4- + 5 H2O2 + 6 H+ 5 O2 + 2 Mn2+ + 8 H2O

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Azide or cyanide ions form very stable complexes and inactivate enzymes

containing ferric ions, but have only a small effect on ferrous ion-containing enzymes.

Cytochromes, catalase and peroxides contain the ferric form and, therefore, are

strongly inhibited by azide or cyanide.

2. MATERIAL.

* Crystallizer or plastic bucket * Ice

* 3 erlenmeyers (25 - 50 mL)

* Pipettes: 110 mL, 22 mL, 21mL, 10.5 mL

* Sterile lancet

* Graduated cylinders (50 mL ) * Burettes (25 or 50 mL )

* Pipette pumps

3. SOLUTIONS OR REAGENTS.

* Blood

* 20 mM sodium phosphate buffer, pH 7.0

* H2O2 (aprox. 50 mM) in 20 mM phosphate buffer (pH 7.0)

* 5x10-5 M sodium azide

* 2.5 mM KMnO4

* 6 N H2SO4

4. METHOD.

Fresh blood, in a dilution of 1:500, is used as source of enzyme. To do this 25

mL of cold distilled water is placed in a 50 mL Erlenmeyer that is in the plastic bucket

with cold water and ice. Two or three drops of blood are obtained from your fingertip

puncturing with a sterile lancet. Blood is released into the Erlenmeyer containing the

cold water. Shake the Erlenmeyer to obtain a homogeneous mixture. Keep cold diluted

blood along throughout the experiment to minimise heat inactivation.

Small erlenmeyers are prepared as indicated in the following table, by adding

the quantities for each one of them. The reaction must be carried out in cold, with

the ice bath. After each addition the Erlenmeyer flask must shake well.

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THE DETERMINATIONS MUST BE MADE DUPLICATE. (TIP: It is preferable to

prepare the two Erlenmeyers of the same number at the same time, and when the

reaction has been stopped then starting with another number).

To stop the reaction, after 5 minutes of the enzyme reacting to the cold, add 2

mL of 6 N H2SO4 to the corresponding Erlenmeyer flask.

Number 1 is the blank (control) of the reaction and represents the total amount

of H2O2 added to each flask. In this case, the addition of 2 mL of 6 N H2SO4 precedes

the addition of the enzyme and it is not necessary that the Erlenmeyer flask is held

5

min. in cold since no reaction occurs. This can be titrated directly.

The Erlenmeyer n º 3 corresponds to the effect of temperature on the enzyme

activity. Therefore it must be put into a test tube about two millilitres of catalase and

heated in water-bath at 50 º C around 5 to 10 minutes. Then, when you h ave to put

the corresponding quantity of " heated catalase ", you take catalase from this tube.

Nº erlenmeyer 1 (control) 2 3 (temp.) 4 (inhib.)

Phosphate buffer, pH 7.0 (mL) 10 10 10 10

H2O2 (mL) 2 2 2 2

Water (mL) 1 1 1 0.5

Azide 5x10-5 M (mL) -- -- -- 0.5

Catalase (mL) 0.5 0.5 -- 0.5

Heated catalase (mL -- -- 0.5 --

The amount of H2O2 that has not been decomposed with catalase will be stable

in acid solution at least half an hour. During this time, complete the titration of

peroxide with 2.5 mM KMnO4. The first drop of permanganate in excess over the

amount required for the oxidation of peroxide puts the solution pink. This is the end

point.

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5. CALCULATION OF ENZYME ACTIVITY.

The activity of catalase can be expressed as µmoles of H2O2 decomposed by the

action of the enzyme in 5 minutes at 0 º C per millilitre of blood:

μmols H2O2 decomposed x 1000 Enzyme activity =

5

To do the calculations, the number of µmoles of H2O2 found in erlenmeyer No.

1 is the zero time (control). The stoichiometry of the decomposition of H2O2 by

permanganate indicates that for every 2 moles of MnO4-added decompose 5 moles of

H2O2.

The factor is 2.5. To calculate the remaining μmoles of H2O2, it should be

multiplied by 2.5, the µmoles of MnO4- added. Subtracting the amount of H2O2 no

decomposed from the volume of H2O2 present initially (Erlenmeyer flask No. 1), we

obtain the µmoles of peroxide decomposed broken down.

As has been done a 1:500 dilution of blood and have been taken aliquots of 0.5

mL for the experiments, the µmoles of H2O2 broken down by catalase must be

multiplied by 1000 to get the enzymatic activity of catalase, according to the definition

given above.

6. QUESTIONS

1. Complete the following table:

1 2 3 4

2.5 mM KMnO4 used (mL)

H2O2 without reacting (μmols)

H2O2 decomposed (μmols)

Enzymatic activity (μmols/mL.min)

Inhibition percentage (%)

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2. What metabolic process is involved in the production of hydrogen peroxide

in the cells? (Check a manual of Biochemistry).

3. What cellular organelle produces hydrogen peroxide?

4. Why should you keep the catalase on ice during the development of practical

exercises?

5. What is the role of sulphuric acid in the practice?

6. What happens to the catalase, at the molecular level, when heated in the

bath at 50 ° C?

7. Explain how azide affects the activity of the enzyme.

18

QUANTITATIVE ESTIMATION OF PROTEINS BY THE BIURET METHOD

1. PRINCIPLES.

Many biochemical experiments involve the measurement of a compound or

group of compounds present in a complex mixture. Probably the most widely used

method for determining the concentration of biochemical compounds is colourimetry,

which makes use of the property that when white light passes through a coloured

solution, some wavelengths are absorbed are absorbed more than others. Many

compounds are not themselves coloured, but can be made to absorb light in the visible

region by reaction with suitable reagents. The colour intensity is proportional to the

concentration of compound (Lambert‐Beer law: A = ε cl).

In colourimetric methods, it is necessary to build a calibration curve using a

standard protein solution of known concentration, representing absorbance versus

concentration. If the Lambert-Beer law is obeyed and ‘l’ is kept constant, then a plot of

extinction against concentration gives a straight line passing through the origin;

whereas a plot of % transmittance against concentration gives a negative exponential

curve. When the line is obtained, the values obtained in the problem solution are

interpolated to obtain the concentration Lowry method based on the reduction of Cu2+

to Cu+ for development of colour.

BIURET METHOD. Proteins and peptides, due to the peptide bonds, give a

coordination complex between copper ions and CO and NH groups of the peptide bond

when they are in an alkaline solution. This complex, violet, gives a maximum

absorption at 545 nm.

The name of the reaction proceeds the colour of the compound formed by

condensation of two molecules of urea and ammonia removal:

BIURET REACTION

BIURET

UREA

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If a strongly alkaline solution of cupric sulphate (CuSO4) is added to a protein

solution a complex is formed between the cupric ion and the peptide bonds, with the

appearance of a violet-purple coloured complex, which has an absorption maximum at

545 nm.

The most important characteristics of the reaction are:

• It is applied from the tetrapeptides to all peptides and proteins.

• The determination range is 1 to 6 mg / mL.

• It does not depend on the amino acid composition.

• Some compounds (NH4 +, TRIS, etc.) give the reaction.

The reaction is not absolutely specific for peptide bonds, since any compound

containing two carbonyl groups linked through nitrogen or carbon atom will give a

positive result.

violet-purple Complex protein - Cu (II)

2. MATERIAL.

* 1 Graduated cylinders (50 mL)

* 1 pipette (2 mL), 1 pipette (5 mL), 1 pipette (0.5 mL) and 2 pipette (1 mL)

* 1 beaker (100 mL)

* Glass test tubes and 2 racks

* Water baths at 37º C and 50º C

* Colorimeter and spectrophotometer

20

3. SOLUTIONS AND REAGENTS.

* Standard solutions of 2.5 mg/mL bovine serum albumin.

* Problem solution of protein.

* Biuret Reagent: Containing CuSO4.5H2O, potassium sodium tartrate and

NaOH.

4. METHOD.

Prepare nine test tubes as follows. DO ALL THE METHOD IN DUPLICATE:

Nº tube 1 2 3 4 5 6 7 8 9

Distilled water

2 1.6 1.2 0.8 0.4 0 1.5 1 0.5

Albumin 2.5 mg/mL

0 0.4 0.8 1.2 1.6 2 -- -- --

Problem -- -- -- -- -- -- 0.5 1 1.5

Add 0.9 mL of solution A to each tube, shake the mixture and incubate at 50 ° C

for 10 minutes. Allow to cool at room temperature and add 0.1 mL of solution B under

stirring and allowing the reaction occurs during 10 minutes at room temperature. After

this time add 3 mL of solution C, rapidly with immediate mixing and incubating at 50 °C

for 10 minutes. Cool at room temperature and read the absorbance in a colorimeter at

650 nm against the test tube nº 1 (the blank).

6. CUESTIONES.

1. Does the standard curve for each method, plot absorbance against the

concentrations of albumin standard?

2. Calculate the protein concentration in the problem tubes: tubes 7,8 and 9.

Should they be equal?

3. Why is it necessary to stain the protein to determine the protein concentration?

4. Considering the Law of Lambert-Beer, what is the slope of the calibration curve?

What are the units?

5. Write the equation of the calibration curve obtained.

6. What is the protein concentration of the original problem?

21

KINETIC STUDY OF POLYPHENOLOXIDASE

1. PRINCIPLES.

The texts of Experimental Biochemistry are an excellent source of Enzymology

experiments. Most allow students to obtain extracts of an enzyme, to assay its

presence in the solution and to study the enzyme kinetics. Few experiments offer the

opportunity for students to apply these techniques to the study of an enzyme. In this

case we have chosen the enzyme polyphenoloxidase or tyrosinase, by studying its

extraction, activity and kinetics. This enzyme was chosen for several reasons: 1) it can

be readily extracted from various plant sources, such as mushrooms, bananas and

potatoes, 2) there is a simple spectrophotometric assay, 3) the metal cofactor allows

inhibition studies, 4) it is important in the chemistry of the food, pigment and clinical

medicine.

The enzyme polyphenoloxidase occurs in both plant and animal cells. Activity in

the presence of oxygen is responsible for the browning of cut or damaged plants.

Tyrosinase from mammalian cells has a specific role in the metabolism of tyrosine. This

enzyme catalyses the conversion of DOPA (3,4-dihydroxyphenylalanine) to the

red-coloured dopachrome, according to the reaction:

In experimental conditions, the reaction follows Michaelis-Menten kinetics.

This sequence of reactions is located in the melanocytes, whose final product is the

production of melanin, substances that give colour to the skin, hair and eyes.

Irradiation of the skin by UV rays of sun activates the tyrosinase, thereby causing the

production of melanins.

Tyrosinase from plant and animal is a copper-containing enzyme which can be

complex by several chelating agents, inactivating the enzyme. Compounds such as

22

phenylthiourea, diethyldithiocarbamate, azide, cyanide or cysteine may are used as

inhibitors.

In this experiment, the polyphenoloxidase extract is prepared from potatoes.

Although various methods of assay are possible, the most convenient involves the

oxidation, enzyme-cataly sed, of 3,4-dihydroxyphenylalanine (DOPA) to

2,3-dihydroindole-5,6-quinone. One can observe the initial rate of formation of the

quinone by the change in absorbance at 475 nm.

Sections a) and b) in the experimental part describe the preparation of the

enzyme and the general assay procedure. Necessary details are given in these two

sections. It may be applied to sections c) and d) where it is studied the effect of

enzyme concentration and the effect of inhibition.

2. MATERIAL.

* Volumetric glass cylinder (50 mL) * 2 Beakers (50 mL and 100 mL)

* Knife * Mortar

* Funnel and tripod * Test tubes and rack

* Colorimeter or spectrophotometer * Pipettes: 1 0.5mL, 3 1mL, 2 5

* Ice and container to keep

3. SOLUTIONS AND REAGENTS.

* Potatoes

* 20 mM Sodium phosphate buffer, pH 7.0.

* DL-ß-3,4 dihydroxyphenilalanine (DOPA) 2 mg/mL dissolved in phosphate buffer

(prepared in the day).

* Inhibitor: 5 mM sodium azide.

4. METHOD.

a) Isolation:

Peel a potato and cut into small pieces. Weigh about 10 grams of potato

quickly, they are crushed in the mortar and then mixed with 50 mL of phosphate

buffer. The mixture is homogenised for one minute and the homogenate is filtered

23

through a paper filter. The extract was prepared immediately before use and stored in

an ice bath during the experiment.

b) Determination of kinetic parameters Km and Vmáx:

Dispose four test tubes in a rack and add buffer and Dopa according to the

following scheme (amounts are in ml):

Nº tube 1 2 3 4

Buffer 2.4 1.9 0.9 0

DOPA 0.5 1 2 2.9

The blank is made with 3 mL of phosphate buffer.

Then, add to the first tube 0.1 ml of extract, mix by inversion and pour quickly

the contents into the cuvette and place the cuvette in the spectrophotometer or

colorimeter, reading the absorbance change at 475 nm for 2 minutes, every 10-15

seconds. This process is repeated with all tubes. The final volume of the reaction

medium is always 3 mL.

c) Effect of enzyme concentration on the activity:

Before considering a valid assay it should be demonstrated that the reaction

rate is directly proportional to the amount of enzyme put in the assay, so this section is

performed to corroborate it.

The procedure is described in section b) by adding the extract, ultimately, just

in time to measure. The blank for adjusting the spectrophotometer is phosphate

buffer, as the previous section, and the wavelength the same as before.

The final reaction mixture should contain the following (in mL):

Nº tube 1 2 3

Buffer 0.9 0.8 0.6

DOPA 2 2 2

Extract 0.1 0.2 0.4

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d) Inhibition studies:

The evidence can be investigated that a metal ion is involved in the enzymatic

reaction, by observing the effect of chelating agents on the enzymatic activity. Copper

chelating agents, such as those mentioned above, are potent inhibitors of tyrosinase or

polyphenoloxidase activity. For this practice we use azide as an inhibitor.

To investigate this effect, a series of tubes are prepared using standard

procedures:

Nº tube 1 2 3

Buffer 0.9 0.5 0.1

DOPA 2 2 2

Inhibitor (azide) 0 0.4 0.8

Extract 0.1 0.1 0.1

5. QUESTIONS.

1. Calculate the initial rates of reaction in each assay by plotting the absorbance

values versus time.

2. Calculate the values of Km and Vmax using the Lineweaver-Burk. In order to

make the plot, previously fill in the following table:

3. Do the plot of the enzymatic activity based on the amount of enzyme.

Previously fill in the following table:

Nº tube v0 (min-1) [Dopa] (mg/mL) 1/v0 (min) 1/[S] (mg/mL)-1

1

2

3

4

Nº tube v0 (min-1) Enzyme volume (mL)

1

2

3

25

4. After the study of the effect of enzyme concentration on the activity, what

conclusions can be inferred from the plot of the enzymatic activity against the volume

of extract?

5. What reaction does polyphenoloxidase catalyse? What is the substrate of the

reaction?

6. Why is the potato extract kept in ice?

7. Why does azide acts as an inhibitor of polyphenoloxidase?

8. Calculate the percentage of inhibition in the presence of different

concentrations of azide, completing the following table:

Nº tube v0 (min-1) [inhibitor] (mM) % inhibition

1

2

3

26

ISOLATION OF DNA FROM HALOPHILIC ARCHAEA. AGAROSE ELECTROPHORESIS.

1. PRINCIPLES.

Nucleic acids, due to its macromolecular character, are rather difficult to

separate from proteins and polysaccharides by gentle methods, and drastic treatments

profoundly alter its structure and its characteristics. Cells from halophilic Archaea need

high salt concentrations to maintain cell integrity. This fact has been used to isolate

their DNA as they can be easily lysed when are treated with water. The cells are

resuspended in water, and the lysate was extracted with phenol. Phenol and water are

immiscible; the proteins are extracted into the phenol phase and separated from the

nucleic acids which remain in the aqueous phase. Adding two volumes of ethanol to

the aqueous phase, the nucleic acids of high molecular weight precipitate as a white

fibrous material. If precipitation occurs as a colourless gelatinous material indicates

that there is still protein bound to nucleic acids.

The concentration of DNA obtained can be determined using the absorbance

(or optical density) at 260 nm. A solution in water, of DNA of 1 mg / mL, produced an

A260 of 20.

When DNA is prepared it must be of high molecular weight and should not be

broken into small fragments. The integrity of DNA can be checked, for each DNA

sample, performing an electrophoresis on an agarose gel 0.8%.

2. MATERIAL.

* Water bath at 65º C

* Eppendorf tubes

* Microcentrifuge

* “Pescador” de DNA

* Electrophoresis equipment

* Micropipettes.

3. SOLUTIONS.

* Ultrapure water (MilliQ) * Phenol (cause burns)

* Absolute Ethanol

* Agarose in TAE (Tris - Acetate- EDTA) buffer

* Red Safe * Dye solution

27

4. METHOD.

To avoid contamination with nucleases, the extraction of DNA must be made

with gloves. All material should be autoclaved (sterilised). Treatments must be made

with care to avoid fragmenting the DNA as little as possible and obtain DNA of high

molecular weight.

4.1 Extraction:

Cells from a fresh culture of halophilic Archaea (Haloferax mediterranei) are

harvested to remove all culture media. Once we have the cell pellet "dry", cells are

lysed by adding 200 μL ultrapure water to the cells precipitate. After adding an equal

volume of phenol (saturated with Tris buffer), mix by inversion and the mixture is

incubated at 65 ° C for 10 minutes. Subsequently, the phases were separated by

centrifugation at room temperature.

Transfer the aqueous phase (top) to a clean eppendorf tube, adding two

volumes (400 μL) of absolute ethanol and stir gently to mix. The precipitated DNA

should be visible at this stage. The precipitate is "fishy" or spin. Redissolved in 100 μL

ultrapure water and incubate at 37 ° C for a time.

4.2. Agarose electrophoresis:

4.2.1. Preparation of agarose gel: Weigh 0.32 g in 200 mL Erlenmeyer flask, add

40 mL of buffer 1xTAE. Heat in microwave until the agarose is dissolved. When the

agarose has cooled, without reaching solidification, 2 µL of Red Safe is added and

placed in the tray of the system; the comb is placed to make the streets and allowed to

solidify.

4.2.2 Electrophoresis: Fill the tank with 1xTAE electrophoresis buffer; the gel is

placed in the tank and the gel should be covered with buffer at least 1 cm. To prepare

the DNA samples to load into the gel, 15 mL of DNA and 3 L of dye are pipetted.

Samples are loaded on the wells of the gel, in one of the wells we can load known

molecular weight markers.

28

Electrophoresis is performed by applying 120 V (50 mA). The samples run from

the negative electrode (cathode) to positive (anode). When electrophoresis finishes,

the DNA can be visualised by ultraviolet light, WITH UV PROTECTION GOGGLES.

5. QUESTIONS.

1. When is it considered that the DNA obtained is of high molecular weight,

qualitatively?

2. What characteristic is observed to see if DNA is broken?

3. How do you calculate the concentration of DNA obtained, qualitatively?

4. How do you calculate the concentration of DNA obtained, quantitatively?

5. In what step of the isolation of nucleic acids is DNA visible? Why is that?

6. Why do we see fluorescence when the DNA is viewed with the transilluminator?

7. The method used for isolating DNA from Haloferax mediterranei, could be

used it to isolate nucleic acids from any microorganisms? Explain your answer.