BIOCHEMISTRY

SCBT 3114

PRACTICAL MANUAL

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Department of Biotechnology,

Faculty of Applied Sciences,

NAME : ___________________________________________________________________

MATRIC NO : ___________________________________ BATCH : ____________________

RULES OF CONDUCT AND GENERAL SAFETY

Anyone who chooses to disregard these rules or exhibits carelessness that endangers others may be subject to immediate dismissal from the laboratory. If doubt arises as to the procedure involved, consult your lecturer or scientific officer. Each student is responsible for the observance of the following rules:

1. Place all extra clothing, unnecessary books, purses, backpacks and paraphernalia in an appropriate place. The laboratory work area must be kept free of articles not actually in use.

2. Eating, drinking and smoking are forbidden at all times in the laboratory.3. Return all equipment to their proper places.4. Wear covered shoes when working in the laboratory. 5. Do not place anything in your mouth while in the laboratory. This includes pencils,

food and fingers. Learn to keep your hands away from your mouth and eyes.6. Long hair should be tied back to minimize fire hazard and your own safety.7. Note where all the specific safety features are located in the laboratory. These would

include the fire extinguisher, the safety shower, the fire blanket, the eye wash station, the first aid kit and emergency exit.

All laboratory work can be done more effectively and efficiently if the subject matter is understood before coming to the laboratory. To accomplish this, read the experiment several times before the laboratory begins. Know how each exercise is to be done and what principles it is intended to convey. This will save you much time and effort during the actual laboratory period.

Learning Outcomes

You will be evaluated on the following Programme Learning Outcomes (PO) :

Programme Learning Outcomes

PO1 Ability to demonstrate a comprehensive understanding of Biotechnology

PO2 Ability to operate and maintain basic Biotechnology equipment

PO4 Ability to analyse, synthesise and integrate knowledge and information

This PO will be commented but NOT evaluated :Programme Learning Outcomes

PO3 Ability to communicate and demonstrate interpersonal skills

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Assessment Criteria

PO1 : Ability to demonstrate a comprehensive understanding of Biotechnology This is to evaluate students on the knowledge aspect of the practical work. It will be based on the submitted written report of a particular laboratory practical.

Marks Descriptors

PO1

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0 The student does not reach a standard described by any of the descriptors given below

1-2 The student recalls some scientific ideas and concepts and applies these to solve simple problems

3-4 The student explains scientific ideas and concepts and applies scientific understanding to solve problems in familiar situations. The student analyses scientific information by identifying parts, relationships or causes. The student provides an explanation that shows understanding.

5-6 The student explains scientific ideas and concepts and applies scientific understanding to solve problems in familiar and unfamiliar situations. The student analyses and evaluates scientific information by making scientifically supported judgments about the information, the validity of the ideas or the quality of the work.

PO2 : Ability to operate and maintain basic Biotechnology equipmentThis is to evaluate students on the practical skills. It will be based on the performance of students during the practical session.

Marks Descriptors

PO2

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0 The student does not reach a standard described by any of the descriptors given below

1-2 The student requires guidance and supervision when using laboratory equipment. The student can work safely and cooperate with others but may need reminders.

3-4 The student uses most equipment competently but might require occasional guidance; on most occasions pay attention to safety and works responsibly with the living and non-living environment. The student generally cooperates well with other students

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5-6 The student works largely independently; uses equipment with precision and skill; pays close attention to safety and deals responsibly with the living and non-living environment. The student consistently works effectively as part of a team, collaborating with others and respecting their views.

PO4 - Ability to analyse, synthesise and integrate knowledge and informationThis is to evaluate students on the data analysis, results presentation, discussion and conclusion. It will be based on the submitted written report of a particular laboratory practical.

Marks Descriptors

PO4

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0 The student does not reach a standard described by any of the descriptors given below.

1-2 The student organizes and presents data using simple numerical or diagrammatic forms and draws an obvious conclusion.

3-4 The student organizes and transforms data into numerical and diagrammatic forms and presents it using appropriate communication modes. The student draws a conclusion consistent with the data.

5-6 The student organizes and transforms data into numerical and diagrammatic forms and presents it logically and clearly, using appropriate communication modes. The student explains trends, patterns or relationships in the data, comments on the reliability of the data, draws a clear conclusion based on the correct interpretation of the data, and explains it using scientific reasoning.

PO3 - Communicate and demonstrate interpersonal skillsThis is to evaluate students on the presentation of the lab reports such as format, spelling, etc. It will be based on the submitted written report of a particular laboratory practical.

Levels Descriptors

Level 0 The student does not reach a standard described by any of the descriptors given below.

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PO3

Level 1 The student attempts to communicate scientific information using some scientific language. The student presents some of the information in an appropriate form using some symbolic or visual representation when appropriate. The student attempts to acknowledge sources of information but this is inaccurate.

Level 2 The student communicates scientific information using scientific language. The student presents most of the information appropriately using symbolic and / or visual representation according to the task. The student acknowledges sources of information with occasional errors.

Level 3 The student communicates scientific information effectively using scientific language correctly. The student presents all the information appropriately using symbolic and / or visual representation accurately according to the task. The student acknowledges sources of information appropriately.

PRACTICAL 1Spectrophotometry and Benedict’s test for sugars

Objective

1. To understand the principle and how to use a spectrophotometer.2. To determine the lambda maximum ( max) of the given solution and its importance3. To perform Benedict’s qualitative test for the determination of reducing and non-

reducing sugars

IntroductionBeer-Lambert Law If a monochromatic light of initial intensity Io passes through a solution, some of the light may be absorbed. Thus, the transmitted light I is less than Io. The ratio of intensities I/Io, are called transmittance and are dependent on several factors: 1. If the concentration [c], of the absorbing solution increases, the transmittance will decrease. 2. If the wavelength [I], that the light must travel through increases, then the transmittance will decrease. 3. If the nature of substance changes or another substance that absorbs more strongly is used, then the transmittance will change. The nature of the substance is reflected in e, the extinction coefficient. log I/Io = e lc l Io I

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Incident beam Emergent

beam

Where Io = intensity of incident light I = intensity of transmitted light e = extinction coefficient l = path length through solution c= concentration of absorbing solution log I/ Io is called the absorbance (A) where A = e l c The equation above is called the Beer-Lambert law. When monochromatic light passes through a solution the intensity of the light transmitted decreases exponentially With increasing path length (Lambert law)With increasing concentration of the absorbing substances (Beer’s law)If a substance obeys the Beer-Lambert law, then a plot of A vs. c is linear. Note that the Law is not obeyed at high concentration.

Determination of lambda maximum ( max) for a given solution

Principle

When measuring the absorbances of several samples that have different concentrations, it

is important to identify an appropriate wavelength at which to take measurements. This

wavelength is the one at which the samples absorb the most light and is called lambda

max, since the Greek letter ‘ ’ is used to symbolize wavelength. The spectrophotometer

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Absorbing soln

conc ‘c’

1. Light source2. Wavelength

Selection3. Sample

chamber4. Detectors 5. Meters or

Recorders

A

Concentration

or a scanning spectrophotometer can be used to help generate a plot of absorbance versus

wavelength, which can then be used to find lambda max.

Materials/ Apparatus

1. Colored Solution to measure the optical density at various wavelength.2. Spectrophotometers

3. Cuvets

4. Pipettes

Procedure

To find lambda max, set the wavelength on the spectrophotometer to 400 nm and

calibrate the maximum and zero absorbance using the reference solution. Insert the

sample solution and record its absorbance at this wavelength. Increase the wavelength in

20 nm increments and record the absorbance at each wavelength. Take measurements

from 400 nm up to 600 nm.

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Remember to recalibrate the maximum and zero absorbance each time you change the

wavelength. Record the values and plot a graph of absorbance versus wavelength as

shown below and calculate the lambda max.

Procedure1. Switch on the spectrophotometer for a while and then set the appropriate wavelength

2. Cuvets should be clean and dry when used. The outside of each cuvet should be wiped clean before it is placed in the instrument.

3. Care should be taken to position the cuvet in the spectrophotometer with the same side facing the light source each time. A mark may be made near the top of the cuvet to guide the alignment.

4. The solution in each cuvet should be free of air bubbles. Slight tapping will remove air bubbles from the tube

5. Set the instrument to measure the absorbance

6 First read the tube marked ‘Blank’ and zero the spectrophotometer with the blank (autozero)

A blank solution is either water or a reagent blankThe primary purpose of the blank is to cancel any absorbance arising from the reagents. Ideally the blank is prepared in the same fashion as the test sample to be analyzed but it doesn’t contain any sample solution

7. The cuvet well should be covered before each reading is made. Light from windows or overhead lights can cause marked errors in readings.

No. Wavelength (nm) Optical density

1. 4002. 4203. 4404. 4605. 4806. 5007. 5208. 5409. 56010 580

11 600

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8. Read the standards and then followed by the test samples.

9. Write out the optical density or absorbance of each standard and test sample and then calculate the concentration of the sample using the formula mentioned below.

Limitations of the Beer-Lambert law

The linearity of the Beer-Lambert law is limited by chemical and instrumental factors.

Causes of nonlinearity include:

deviations in absorptivity coefficients at high concentrations (>0.01M) due to

electrostatic interactions between molecules in close proximity

scattering of light due to particulates in the sample

fluoresecence or phosphorescence of the sample

changes in refractive index at high analyte concentration

shifts in chemical equilibria as a function of concentration

non-monochromatic radiation, deviations can be minimized by using a relatively

flat part of the absorption spectrum such as the maximum of an absorption band

stray light

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Qualitative estimation of glucose by Benedict test

Principle

Carbohydrates that contain aldehydes or hydroxymethyl ketones can be oxidized by

Cu(II) ion and are classified as reducing sugars. They reduce the Cu(II) ion to Cu(I).

Benedict reagent is composed of copper sulphate, sodium carbonate, and sodium citrate

(pH 10.5). The citrate will form soluble complex ions with Cu++, preventing the

precipitation of CuCO3 in alkaline solutions.

Composition of Benedict reagent

Solution A: Dissolve 1.73 g trisodium citrate (dihydrate) and 1.0 g anhydrous sodium

carbonate in 8 mL of warm distilled H2O.

Solution B: Dissolve copper sulphate (pentahydrate) (1.73 g) separately in 20 mL of

distilled H2O.

Immediately before using, prepare Benedict reagent by mixing 0.8 mL of Solution A with

0.2 mL of Solution B.

To test for reducing sugars, add 0.2 mL of a 1% carbohydrate solution to 1 mL of

Benedict reagent and heat in a boiling water bath for 5 minutes. Remove the tubes from

the heat and allow them to cool. A brick-red precipitate indicates a positive test for

reducing sugars. Alkaline solutions of copper are reduced by sugars having a free

aldehyde or ketone group, with the formation of colored cuprous oxide.

Sugar Observation Comments

A. Glucose

B. Fructose

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C. Sucrose

D. Starch

E. Unknown sugar

* Report need to be submitted one week after the practical.

Questions:

PO1 : Ability to demonstrate a comprehensive understanding of Biotechnology

1. What is the range of visible range wavelength and name the light source used in the

visible range?

2. What is the relationship between absorbance and concentration of a solution and if

the concentration of the solute is high in a solution how to we measure the absorbance

using a spectrophotometer. Give reasons for your answer.

3. What are the main components or part of a spectrophotometer and its functions?

4. What does lambda max for a solution tells us?

5. Name some sources of starch and also name what type of reducing sugars are present

in milk, table sugar, fruits.

PO4 - Ability to analyse, synthesise and integrate knowledge and information

1. What is the reaction that takes place when you add Benedict’s solution to a sugar

solution?

2. Is cellulose a reducing or non-reducing sugar? Can humans digest cellulose? Explain

your answer

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PRACTICAL 2: Titration Curve of amino acid

Aim

The aim of this practical class is to demonstrate how amino acids in solution can behave as:(i) proton donors (acids), (ii) proton acceptors (bases) and (iii) buffers (mixtures of both weak acids and bases).

Important notes• Please revise the general structure of amino acids and pH and buffers beforehand.• Absence from a practical will result in a zero marks

Objective

After successfully completing this practical class, students should be able to:1. Use pH meters and pH electrodes, obtain titration curves and from these graphs

estimate pK (strictly pKa) values for both diprotic and triprotic acids.2. Indicate, on each titration curve, the location of (a) equivalence points and (b) regions

of maximum buffering.3. Demonstrate their understanding of which ionisable groups of amino acids are able to

contribute to the buffering properties of proteins.

IntroductionThese experiments are designed to demonstrate the ionic condition of amino acids

as a function of pH, and how to estimate their pKa values. Glycine is described as ‘diprotic’ because there are two protons which may be removed from the acidic (fully protonated) ionic form of the molecule. Other ‘triprotic’ amino acids have ionisable side chains which, in the acid form, possess a third removable proton.The addition of 25 ml NaOH to 25 ml equimolar glycine in the acidic (most fully protonated) form, will remove the carboxyl protons and convert the cation to the zwitterions

HOOC.CH2.NH3+ + NaOH -OOC. CH2.NH3

+ + H2O + Na+

cation zwitterionThe proton is initially removed from the carboxyl group rather than the (positively charged) amino group, because the latter has a greater affinity for protons.Further addition of more NaOH will remove the amino group protons, as the zwitterions is converted to the anion-OOC. CH2.NH3

+ + NaOH -OOC. CH2.NH2 + H2O + Na+

zwitterion anionIn the case of triprotic amino acids, an additional equation is required, to demonstrate how a third proton may be removed from the side chain (R-group) by the further addition of NaOH.

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The order of proton dissociation depends on the acidity of the proton: that which is most acidic (lower pKa) will dissociate first. Consequently, the H+ on the α-COOH group (pKa1) will dissociate before that on the α-NH3 group (pKa2). The titration curve for this process looks similar to the following:

Procedure

1. Follow the instructions for calibration of the pH meter by the SO. (It is usual to calibrate the pH meters at the start of any series of pH measurements, using standard buffers of known composition and pH value.) Please do not adjust the pH meter controls, as this will affect the calibration.

2. Pipette 25 ml of the amino acid solution (50 mM) into a clean, 150 ml capacity beaker, using a glass bulb pipette and safety bulb. Set up the apparatus as shown below:

3. Remove the glass electrode from the beaker of distilled water and rinse briefly with distilled water from a plastic wash bottle.

4. Arrange the position of the beaker containing the 25 mls of the amino acid solution so that the bulb of the glass electrode is immersed in the solution. Measure the pH of the solution, from the reading on the pH meter scale.

5. Record this initial pH value, and all pH values subsequently in the ‘Results’ section. These will be used later to plot the values on graph paper, to yield a titration curve.

6. Be careful while pouring sodium hydroxide in case of splashes. Make sure that the burette is filled to the 0 ml mark with sodium hydroxide solution (50imM) using a

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clean beaker and plastic funnel to aid filling the burette. Make 0.5 ml additions of the sodium hydroxide from the burette, mixing the solution in the beaker carefully, and recording the pH after each addition. (Wait for the meter reading to stabilise, before recording the pH).

7. Continue the experiment by making further additions of 0.5 ml of NaOH solution, until 50 ml (for triprotic acids) of the base has been added.

8. On graph paper, plot your titration curve as pH (y axis) against mls NaOH (x axis) using the largest scales that are appropriate for the size of the paper. Join the points (pencil only) to form a smooth curve or Using Microsoft Excel, construct your titration curve plotting pH versus mL of base added to the amino acid solution.

9. On your curve, designate the buffer region(s), pKa(s), and the amino acid’s pI.

10. On each graph, draw the ionic structure of the amino acid present at the beginning of the titration, and also at each equivalence point (three in the case of triprotic amino acids). *Report need to be submitted one week after the practical.

ResultsName of the amino acid titrated: ___________Record the initial pH value of the amino acid solution, and then the pH values read as the NaOH is added in the course of the titration.

Vol. of NaOH(ml)

pH Vol. of NaOH(ml)

pH

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Vol. of NaOH(ml)

pH Vol. of NaOH(ml)

pH

pKa valuesInsert the pKa values that you have found into the table below. State the functional group (α-carboxyl, α-amino or side chain) to which each pKa belongs.

Name of Amino acid

pKa1 group pKa2 group pKa3 group

Questions:

PO1 : Ability to demonstrate a comprehensive understanding of Biotechnology

1. Draw the ionic structure of each amino acid in the form that would be the predominant ionic species present at pH 7.Glycine Aspartic acid

Arginine Histidine

2. Say True or False

A. The amino acid glycinea. is positively charged at pH1.b. is negatively charged at pH12.

B. pKa2 for aspartic acida. is the pKa of the amino group.b. is around pH7.

C. Adding sodium hydroxide to your amino acid solution

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a. increased the pH of the solution.b. increased the concentration of solvated protons (H+ ions) in the solution.

D. Your titration experiment showed that amino acids show maximum bufferingcapacity at pHs around theira. pKa values.b. equivalence points.

PO4 - Ability to analyse, synthesise and integrate knowledge and information

1. Briefly describe how a buffer functions, indicating at which pH it is most effective.

2. Which ionisable groups of amino acids cannot contribute to the buffering properties of proteins?

3. What are the possible sources of error, when estimating pKa values in this practical class?

4. Why must the magnetic stirrer be stopped each time before reading the pH?

5. Explain briefly the principle of pH

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PRACTICAL 3Determination of Maltose in the given sample by colorimetric

assayObjective

1. To become familiar with colorimetry as an analytical tool in biochemistry.2. To establish a standard curve for the determination of maltose and determine the

concentration of maltose in unknown samples.

Principle

This method tests for the presence of free carbonyl group (C=O), the so-called reducing sugars. This involves the oxidation of the aldehyde functional group present in, for example, maltose and the ketone functional group in fructose and the simultaneous reduction of 3,5-dinitrosalicylic acid (DNS) to 3-amino,5-nitrosalicylic acid under alkaline conditions:

Aldehyde group Carboxyl group

3,5-Dinitrosalicylic acid 3-amino 5-nitrosalicylic acid

Maltose can be detected after reacting with 3,5-dinitrosalicylic acid, a component of the Maltose Colour Reagent. The absorbance is read at 540nm.

SAFETY GUIDELINESWhen handling the Maltose Colour Reagent (1% 3,5-dinitrosalicyclic acid, 2M NaOH, 1.06 M sodium potassium tartrate) as this concentration of NaOH is slightly corrosive. Flush your skin or eyes with water if there is contact.

Procedure1. Label six round bottom, 15 ml tubes from ‘1’ to ‘6’for standards and T1 and T2 for

the unknown samples. Add the appropriate volumes of water and maltose, listed in Table 1, to each tube. Add 1.0 ml of Maltose Colour Reagent to each tube. Cover with parafilm and vortex to mix.

Table 1 Unknown

Tube1

(Blank)2 3 4 5 6

T1 T2

Deionized water [ml] 2.0 1.8 1.6 1.4 1.2 1.0 0 0

Maltose (2mg/ml) [ml]

0 0.2 0.4 0.6 0.8 1.0 2.0 2.0

Maltose Color Reagent [ml]

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

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oxidation

reduction

in alkaline medium

2. Place the tubes in a hot plate set at 100oC (or a boiling water bath) for exactly 15 minutes. At this temperature, the 3,5-dinitrosalicylic acid in the Maltose Colour Reagent reacts with maltose to produce darker orange color solution that absorb light at 540nm.

3. Place the tubes on ice until cooled to room temperature. Add 9 ml deionized water (dH2O) to each tube. Cover the tubes with parafilm, and mix the contents by inverting the tubes several times. (Since the tubes are very full, vortexing does not mix the contents very well).

4. Using tube 1 (Reagent Blank) as the blank to zero the spectrophotometer, measure the absorbance of other tubes (2-6) at 540 nm. Record the results in Table 2.

Table 2Tube 1

(Blank)2 3 4 5 6 T1 T2

A540nm

mg of maltose

a. Calculate the mass (mg) of maltose in each sample and record the value in Table 2. Mass (mg) of maltose can be calculated by using the information given in Table 1.

For example,0.2 ml x 2 mg/ml = 0.4 mg maltose

b. Plot the data in table 2 on graph paper, putting the total mass (mg) of maltose on the X- axis and the absorbance on the Y- axis. Using a ruler to draw a straight best- fit line through the origin and the data points.

QUESTIONS:

PO1 : Ability to demonstrate a comprehensive understanding of Biotechnology

1. Name the chemical that is present in the reagent you have used in the lab to estimate the maltose.

2. Name the two reducing groups that are generally present in sugars?

3. What is the relationship between optical density (OD) and absorbance (A)?

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4. What is the importance of reagent blank in the experiment?

5. Write the reaction that takes place when maltose reacts with maltose color reagent

PO4 - Ability to analyse, synthesise and integrate knowledge and information

1. Name a different method for the quantitative estimation of maltose other than the method you have done in the laboratory by which reducing sugars are

2. Is this colorimetric assay carried out in the laboratory qualitative or quantitative method? Give reasons for your answer

3. If you have a stock solution of maltose of 120mg/ml, and if you are using 0.35ml for your assay what is the concentration of maltose in it.

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Practical 4 Determination of protein in the given sample by Bradford colorimetric

assayObjective

1. To construct a standard curve using Bovine serum albumin (BSA) 2. To calculate the amount of unknown protein present in the sample 1 and 2

Introduction

The Bradford assay is very fast and uses about the same amount of protein as the Lowry assay. This procedure is used to measure protein concentration in samples. It is fairly accurate and samples that are out of range can be retested within minutes. The Bradford is recommended for general use, especially for determining protein content of cell fractions and assesing protein concentrations for gel electrophoresis.

Principle

The Bradford assay is a protein determination method that involves the binding of Coomassie Brilliant Blue G-250 dye to proteins (Bradford 1976). The dye exists in three forms: cationic (red), neutral (green), and anionic (blue). Under acidic conditions, the dye is predominantly in the doubly protonated red cationic form (Amax = 470 nm). However, when the dye binds to protein, it is converted to a stable unprotonated blue form (Amax = 595 nm). It is this blue protein-dye form that is detected at 595 nm in the assay using a spectrophotometer or microplate reader. The assay is useful since the extinction coefficient of a dye-albumin complex solution is constant over a 10-fold concentration range.Interference from non-protein compounds is due to their ability to shift the equilibrium levels of the dye among the three colored species. Known sources of interference, such as some detergents, flavonoids, and basic protein buffers, stabilize the green neutral dye species by direct binding or by shifting the pH . Nevertheless, many chemical reagents do not directly affect the development of dye color when used in thestandard protocol.

Materials

1. Bradford reagent contains Coomassie Brilliant Blue G-250 in 95% ethanol and 85% (w/v) phosphoric acid. 2. Bovine Serum albumin (BSA) standard: 2mg of BSA dissolved in 1ml of water. This is stored at 4oC.3. Protein with Unknown concentration4. Clean Test tubes5. Pipetteman and tips

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Table 1

TubeStandard/sample

volume (ml)mg/ml BSA stock soln

Bradford dyeOD reading

Blank 0 1.5ml

Std 1 0.1 0.25mg/ml 1.5ml

Std 2 0.1 0.5mg/ml 1.5ml

Std 3 0.1 0.75mg/ml 1.5ml

Std 4 0.1 1.0mg/ml 1.5ml

Std 5 0.1 1.25mg/ml 1.5ml

Std 6 0.1 1.5mg/ml 1.5ml

Sample1 0.1 1.5ml

Sample 2 0.1 1.5ml

Procedure

1. Label separate clean test tubes for standards and blank as 1 to 6. and for samples as S1 and S2.

2. Add 100l of the appropriate standard BSA solution and samples into the tubes as shown in Table 1.

3. As for the sample tube add 100l of the given sample into the respective tubes.

4. Pipette 1.5ml of 1X Bradford reagent into each tube and vortex

5. Incubate at room temperature for atleast 5 min. (Note:Samples should not be incubated longer than 1 hr at room temperature).

6. Set the spectrophotometer to 595 nm. Zero the instrument with the blank tube. Measure the absorbance of the standards and unknown samples.

Data Analysis

Create a standard curve by plotting the 595 nm values (Y-axis) versus their concentration in μg/ml (X-axis).

Determine the unknown sample concentration using the standard curve. If the samples were diluted, adjust the final concentration of the unknown samples by multiplying by the dilution factor used.

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QUESTIONS:

PO1 : Ability to demonstrate a comprehensive understanding of Biotechnology

1. What does BSA stands for in protein estimation?

2. What is the principle behind Bradford assay

3. Is the calibration curve in the Bradford method straight or crooked/curved? What is the significance of the shape?

PO4 - Ability to analyse, synthesise and integrate knowledge and information

1. Name a different method other than you have done in the laboratory by which proteins are quantitated

2. Can you name a non-colorimetric method by which proteins can be estimated?. What is the principle behind it?

3. Name few food items that are rich in proteins

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Practical 5Determination of catechol oxidase activity from potato

Determination of effect of temperature and inhibitor activity on enzyme activity

Objectives1. To study the enzyme activity of catechol oxidase extracted from potatoes.2. To study the effect of different temperature on the enzyme activity3. To analyze whether EDTA inhibits the catechol oxidase activity by competitive

or noncompetitive inhibition

INTRODUCTIONLiving cells perform a multitude of chemical reactions very rapidly because of the

participation of enzymes. Enzymes are biological catalysts, compounds that speed up a chemical reaction without being used up or altered in the reaction. The material with which the catalyst reacts, called the substrate, is modified during the reaction to form a new product. But because the enzyme itself emerges from the reaction unchanged and ready to bind with another substrate molecule, a small amount of enzyme can alter a relatively enormous amount of substrate.

The active site of an enzyme will bind with the substrate, forming the enzyme-substrate complex. Enzymes are, in part or in whole, proteins and are highly specific in function. Because enzymes lower the energy of activation needed for reactions to take place, they accelerate the rate of reactions. They do not, however, determine the direction in which a reaction will go or its final equilibrium.

Enzyme activity is influenced by many factors. Varying environmental conditions, such as pH or temperature, may change the three-dimensional shape of an enzyme and alter its rate of activity. Specific chemicals may also bind to an enzyme and modify its shape. Chemicals that must bind for the enzyme to be active are called activators. Cofactors are nonprotein substances that usually bind to the active site on the enzyme and are essential for the enzyme to work. Organic cofactors are called coenzymes, but other cofactors may simply be metal ions. Chemicals that shut off enzyme activity are called inhibitors, and their action can be classified as competitive or noncompetitive inhibition.

Principle

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Figure 1: The oxidation of catechol. In the presence of catechol oxidase, catechol is converted to benzoquinone. Hydrogens removed from catechol combine with oxygen to form water.

ProcedureIn this practical, you will be given a 1% catechol solution and catechol oxidase enzyme solution extracted from potatoes. Keep the enzyme on ice to minimize their inactivation. You will be performing an experiment using a potato extract prepared for you.Catechol is a phenolic compound found in many plants. When plant tissues are injured catechol is released and rapidly oxidized by catechol oxidase to benzoquinone. Benzoquinone is toxic to bacteria and thus helps protect the plant from infection. On exposure to air there is a further reaction in which the yellow benzoquinone is converted to dark brown melanin.

SAFETY GUIDELINES*Catechol is a poison! Avoid contact with all solutions. Do not pipette any solutions by mouth. Wash hands thoroughly after each experiment. If a spill occurs, notify the instructor.

Preparation of Potato Extract Weigh 1 hand full of cut potatoes, and place in a blender. Add an equivalent amount of ice-cold distilled water. (For example if your hand full

of potatoes weighs 200g, then add 200ml of water) Blend well. Strain through cheesecloth and be sure to keep extract on ice at all times!

Experiment 1

Catechol Oxidase Activity

Using Table 1, prepare the three experimental tubes. Note that all tubes should contain the same total amount of solution. Do not cross-contaminate pipettes! After each tube is prepared, use your finger to hold a ParafilmTM square securely over the tube mouth and then rotate the tube to mix the contents thoroughly. Use a fresh square for each tube.

Table 1: Contents of three experimental tubes

Tube Distilled Water

1% Catechol

Potato Extract

Color change

A 5.5ml 0.5ml

B 5.5ml 0.5ml

C 5ml 0.5ml 0.5ml

Observe the color change after 20 mins

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Experiment 2

Effect of Temperature on Enzyme ActivityIn this exercise you will determine the effect of temperature on the activity of the

enzyme, catechol oxidase at five different temperatures (ice water, room temperature, 40°C, 60°C and Boiling water).

Use 6 test tubes set up as follows:Table 2:

Conditions/Reagents

Tube1 Tube2 Tube3 Tube4 Tube5 Tube 6

(Control)

Temperature Ice water Room temperature

40oC 60oC 100oC Room temperatu

re

1% Catechol solution

3ml to each tube 3ml

Potato extract solution

3ml to each tube 0.0ml

Cover with a piece of parafilm and mix well.

Incubate the tubes at various temperatures and observe the color change after 20mins against the control.

Experiment 3

Inhibiting the Action of Catechol OxidaseCofactors are inorganic compounds, usually metallic ions, that are a part of the active site of enzymes. Cofactors make the formation of an enzyme-substrate complex possible. Copper is the cofactor for catechol oxidase. This exercise is to determine whether copper is essential for catechol oxidase to produce benzoquinone. EDTA (ethylenediaminetetraacetic acid) binds copper and makes it unavailable for use as a cofactor.

Tube Distilled Water 1% Catechol

Potato Extract

0.5M EDTA Color change

1 5.5ml 0.5ml 1ml

2 5.5ml 0.5ml 1ml

3 5.5ml 1.0ml 0.5ml 1ml

4 5ml 0.5ml 0.5ml 1ml

Observe the color changes in the tubes

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Questions PO1 : Ability to demonstrate a comprehensive understanding of Biotechnology

1. What is the alternative name for catechol oxidase

2. What is the purpose of tube A, tube B and tube C in experiment 1?

3. What is the optimal temperature for catechol oxidase activity? Defend your answer.

4. What temperature produced the lowest activity? Explain, in terms of enzyme function

PO4 - Ability to analyse, synthesise and integrate knowledge and information

1. When, in the fall, you harvest vegetables for prolonged storage in a freezer what is the best way to store them in the fresh form. Can you provide an explanation for this?

2. What is a thermostable enzyme. Name the most common thermostable enzyme that is commonly used in molecular biology

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3. Does EDTA inhibits the catechol oxidase enzyme activity. If so what is its mode of inhibition. Using suitable diagram explain what is the type of inhibition

4. In experiment 3 why there is an addition excess of substrate added

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Practical 6Determination of catechol oxidase activity from potato

Determination of effect of pH on enzyme activity

Objectives1. To study the enzyme activity of catechol oxidase extracted from potatoes.2. To study the effect of different pH on the enzyme activity

IntroductionpH is a measure of the hydrogen ion concentration in a solution. Generally, most enzymes function best at a pH near neutral (pH 6-8). Enzymes are adapted to the environment in which they are active. For example, enzymes that are active during chemical digestion in the stomach function at a lower pH than those active in the small intestine.

Principle

Figure 1: The oxidation of catechol. In the presence of catechol oxidase, catechol is converted to benzoquinone. Hydrogens removed from catechol combine with oxygen to form water.ProcedureUse 6 test tubes set up as follows:Table 1:Conditions/Reagents Tube1 Tube2 Tube3 Tube4 Tube5 Tube 6

pH pH 2 pH 4 pH 6.0 pH 8.0 pH 12.0

1% Catechol solution 3ml to each tube 3ml

Potato extract solution

3ml to each tube 0.0ml

Cover with a piece of parafilm and mix well.

Incubate the tubes at various pH and observe the color change after 20mins against the control.

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QUESTIONS

PO1 : Ability to demonstrate a comprehensive understanding of Biotechnology

1. At what pH does catechol oxidase work best? Give reasons for your answer

2. Name two enzymes that work best in the acid range

3. Name two enzymes that work best in the alkali range

PO4 - Ability to analyse, synthesise and integrate knowledge and information

1. Pepsin is a gastric enzyme does it have an acid or basic optimum pH? What happens to pepsin when it passes into the duodenum?

2. What is the blood pH in humans. What happens when there is an fluctuation in the pH

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