THE ARAB AMERICAN UNIVERSITY

LAB MANUAL
FOR
BIOCHEMISTRY
01021312

2017-2018

PREPARED BY

PROF. BASHAR SAAD

DR. HILAL ZAID
DR. SIBA SHANAK
MS. JAMILA DARAGHMEH
MR. SAID KHASIB

1
 Contents

GENERAL INSTRUCTIONS ................................................................................................................ 3

LABORATORY 1 - BASIC BIOCHEMICAL TECHNIQUES............................................................. 6
A) USE OF PIPETMEN AND BIOCHEMICAL CALCULATION...................................................... 6
B) SPECTROSCOPY AND  MAX DETERMINATION
…………………………………………………………………………..
C) CALCULATIONS, DILUTIONS AND SOLUTION PREPARATION .............................................9
LABORATORY 2 - ANALYSIS OF BIOMOLECULES .................................................................... 17
A) TITRATION CURVES OF AMINO ACIDS .................................................................................. 17
B) THIN LAYER CHROMATOGRAPHY (TLC) ……………………………………………………..21
C) ANALYSIS OF POLY AND MONOMERS ................................................................................. 29
LABORATORY 3 - PROTEIN ANALYSIS ...................................................................................... 41
A) ISOLATION AND PURIFICATION OF PROTEINS.................................................................... 41
B) FRACTIONATION OF PLASMA PROTEINS .............................................................................. 44
C) PHOTOMETRIC DETERMINATION OF PROTEIN CONCENTRATION ............................... 46
D) SDS PAGE ....................................................................................................................................... 49
LABORATORY 4 - ENZYME ACTION ............................................................................................. 56
A) ENZYME KINETICS ANALYSIS OF ALKALINE PHOSPHATASE ......................................... 58
B) INHIBITOR EFFECT ON ALKALINE PHOSPHATASE ACTIVITY ........................................ 62

2
 General Instructions
The following safety and lab instructions should be followed throughout the whole labs.

Laboratory safety

1. Wear a coat to protect your clothing.

2. Do not eat, drink or smoke in the lab.

3. Dangerous, toxic, and flammable chemicals should be handled with care.

4. Wash hands with soap and water whenever you use chemicals or cultures.

5. If you have long hair, tie it back during lab period.

6. Wear eye protectors and if a substance splashes into your eyes, wash them for 5-10

minutes with water.

7. Read labels carefully before using any substance and take only the needed amount.

Laboratory instructions

1. Before each laboratory period, plan your work carefully.

2. Do not miss any lab. In case of sickness, a valid excuse should be presented to the lab

instructor within three days.

3. Be on time for lab. The first part of lab will be used by the laboratory instructor to

introduce the material of the lab. Quizzes for the previous lab can happen.

4. Discard used chemicals and materials into appropriately labeled containers. Some

chemicals can be washed down a sink.

5. Report any accidents such as cuts, burns and spills to your instructor.

6. Leave your laboratory desk clean. Put papers, materials, used slides and coverslips into

appropriate waste containers.

3
Preparing a Laboratory Report

The lab report should be written in a paper format: Abstract, introduction, materials and methods,

results, discussion and conclusion according to the format which follows.

The format that you should follow:

Date_______________________

Name______________________

ID________________________

Title of the Lab_____________________________________________________

Abstract

Introduction

a. Background information
b. Purpose
c. Hypothesis

Materials and methods

Results

a. Graph or table
b. Description of data
c. Answer the questions found in the procedure

Discussion

a. Support hypothesis
b. Explanation
c. Significance of the results

Conclusion

If there are questions, answers for the questions should be presented at the end of each report.

4
 Laboratory 1

1A. Use of pipetmen and Biochemical calculation

Take a cup of distilled water (DW) to the balance. Place an empty small (~ 50ml) cup in the

balance, set the balance record to zero, then pipet out 4 times each of the following volumes:

a1) 500.0 microliters with the 500-5000 microliter micropipetter

a2) 500.0 microliters with the 100-1000 microliter micropipetter

b1) 100.0 microliters with the 100-1000 microliter micropipetter

b2) 100.0 microliters with the 20-200 microliter micropipetter

c1) 50.0 microliters with the 20-200 microliter micropipetter

c2) 50.0 microliters with the 10-100 microliter micropipetter

d1) 10.0 microliters with the 5-50 microliter micropipetter

d2) 10.0 microliters with the 0.5-10 microliter micropipetter

1. Record the weight of water added each time.

2. For each volume taken, calculate the expected water mass according to the following equation:

M=VᵪD. Water density=1g/mL

3. Calculate the average and standard error for the values you got in experiment. Compare the

average of experimental results you obtained in 1 with results in 2. According to the results you

achieve, calculate percentage error according to the following equation:

#𝑒𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡𝑎𝑙 (𝑎𝑣𝑒𝑟𝑎𝑔𝑒) − #𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙

%𝐸𝑟𝑟𝑜𝑟 = | | . 100
#𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙

5
Units of Biochemistry

1.1 International System of Units (SI):

Length: meter

Mass: kilogram

Amount of substance: mole

Time: second

Electric current: ampere

Thermodynamic temperature: kelvin

1.2 Conversion Factor:

A conversion factor is used to change the units of a measured quantity without changing its value.

In one method, the unit bracket method, unit conversion consists of a fraction in which the

numerator is equal to the denominator, but they have different units.

The following set can be interconverted in any unit, and even between units:

k (kilo) 103

m (milli) 10-3

 (micro) 10-6

n (nano) 10-9

p (pigo) 10-12

fg (femto) 10-15

An example of the conversion factor:

If you are asked to convert 10 kg of sugar to the mg unit, then you take the 5 kg aside and check

for conversions between kg and mg:

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 1𝑘𝑔 103 𝑔
1 kg = 103g→( = = 1).
103 𝑔 1𝑘𝑔

1𝑔 103 𝑚𝑔
1g=103 mg→( = = 1).
103 𝑚𝑔 1𝑔

In the above two equations, we use one of the two fractions only where needed, as shown below:

103 𝑔 103 𝑚𝑔
5𝑘𝑔 × × = 5 × 106 𝑚𝑔
1𝑘𝑔 1𝑔

1.2 Mol Units:

Number of moles = number of grams of molecule/molecular weight of molecule

For example: Glucose has a molecular weight of 180g

103 𝑚𝑚𝑜𝑙
# of moles in 10g Glucose = 10g/180g.mol-1 = 0.055 mol = 0.055 𝑚𝑜𝑙 × = 5.5 ×
1𝑚𝑜𝑙

103 𝜇𝑚𝑜𝑙
10 𝑚𝑚𝑜𝑙 => 5.5 × 10 𝑚𝑚𝑜𝑙 × = 5.5 × 104 𝜇𝑚𝑜𝑙 =>
1𝑚𝑚𝑜𝑙

103 𝑛𝑚𝑜𝑙
5.5 × 104 𝜇𝑚𝑜𝑙 × = 5.5 × 107 𝑛𝑚𝑜𝑙
1𝜇𝑚𝑜𝑙

1.3 Concentration Units

The concentration unit is molar. One Molar solution is 1 mol of solute in 1 liter of solution.

1Molar (M) = 1mol/l = 1mmol/ml

1mM = 1mmol/l = 1mol/ml

1M = 1mol/l = 1nmol/ml

For example: 10g Glucose/l= (10g mass/180g.mol-1 MW)/l = 0.055mol/l = 0.055M = 55.5mM =

5.5x 104 M = 5.5 x107 nM

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Questions:
a. How many grams of glucose (C6H12O6) do you need to prepare
1. 5mM glucose in 10ml of solution
2. 5nM glucose in 10ml of solution

b. How many grams of sucrose (C12H22O11) you will use to prepare

1. 10ml of sucrose in 10mM solution
2. 50ml of sucrose in 50nM solution

c. Calculate the concentration (M) of the following solutions

1. 10g serum albumin (MW = 55000) dissolved in 250ml
solution
2. 25ng insulin (MW=5000) dissolved in 1ml solution.

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 1B. Spectroscopy and  max determination
A spectrophotometer is an instrument used for measuring the absorbance of a solution.

A spectrophotometer is commonly used for the measurement of absorbance or transmittance of

solutions at specific wavelengths (λ) that range between 200nm - 2500nm.

Spectrophotometers are among the most widely used research instruments in biology and

biochemistry. For instance,they can be used to determine the density of suspended cells (e.g.

bacteria) or the concentration of a biological entity (e.g., proteins or DNA). The wavelengths at

which most of the biological entities are detected are in the ultraviolet (UV)-visible light range.

Thus, the spectroscopy commonly used in biochemistry labs is called UV-visible spectroscopy.

The ultraviolet (UV) region is from 200 to 400 nm, and the visible portion ranges from 400 to

800 nm. On the other hand, IR spectrophotometer uses light over the infrared (IR) range (800 -

15000 nm) of spectrum.

In visible spectrophotometry, the transmission or the absorption of a certain substance can be

determined by the observed color. For instance, a substance that transmits all visible wavelengths

(i.e., absorbs nothing), appears white in theory. On the other hand, if a substance transmits none

of visible wavelengths (i.e., absorbs light over all visible ranges), then it appears black. If a

solution sample absorbs red light (~700 nm), it appears green because green is the

complementary color of red. The complementary color of orange is blue. Thus, if a substance

absorbs orange (~600 nm), then it transmits blue and appears in blue color…, etc. Visible

spectrophotometers, in practice, use a prism to make it possible for a particular beam of light to

9
pass through a solution sample. Thus a certain range of wavelength is narrowed down (other

wavelengths are filtered out).

Figure 1 shows the basic structure of spectrophotometers. It consists of a light source, a

collimator, a monochromator, a wavelength selector, a cuvette for sample solution, a

pho

toel

ectr

ic

dete

ctor

and a digital display or a meter. Detailed mechanism is described below.

Figure 1. Basic structure of spectrophotometers

A spectrophotometer, in general, consists of two devices; a spectrometer and a photometer. A

spectrometer is a device that produces and disperses light. A photometer indicates the

photoelectric detector that measures the intensity of light.

 Spectrometer: It produces a desired range of wavelength of light. First, a collimator (lens)

transmits a beam of light (photons) that passes through a monochromator (prism). The

monochromator splits the light into several individual component wavelengths of the

spectrum. Then a wavelength selector (slit) transmits only the desired wavelengths, as shown

in Figure 1.

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  Photometer: Once the

desired wavelength of light passes

through the solution of a sample in

cuvette, the photometer detects the

amount of photons that is absorbed

and then sends a signal to a digital display, as shown in Figure 1.

The light intensity of the transmitted light (I) is smaller than that for the incident light (I0).

Figure 2. Incident, absorbed and transmitted light

I/ I0 is known as transmission T.
T = I/I0 = 10-.l.c → %T = Tᵪ100%
T = 10- →A=-logT

The Beer-Lambert law states that: A = lc

A: absorbance of the sample at a particular wavelength (unitless)

: the molar extinction coefficient (also called the molar absorptivity or attenuation coefficient)

for the compound in (M•cm)-1;

c: the molar concentration of the sample (M)

l: the path length of the solution in cm (usually 1 cm in the cuvettes used in the lab).

As it can be seen from the equation above, path length is kept constant for a given experiment.

Also, the molar absorptivity for a chemical is constant, since this value is an intrinsic property of

the single species. As a result, transmission is an exponential function of the concentration of the

11
species. On the other hand, absorbance is a linear function of concentration. Therefore, you may

use the extinction coefficient of an absorbing species (if known) to calculate the absorbance or

the concentration of an unknown solution.

Figure 3. Absorbance vs transmittance curves as a function of concentration

Wavelength at Maximum Absorbance

When you apply the Beer Lambert’s law, you should note to fix the light wavelength before you

plot the absorbance vs concentration of the solute. In fact, each solute has a range of absorbance

values if the wavelength is varied. At one of these wavelengths, called λmax, solute gives the

maximum absorbance. As such, it is recommended to perform the absorbance vs concentration

plot at this wavelength value. Different solutes have different λmax values. As a result, for each

solute, the scan of wavelengths has to be performed before the experiment is set. p-nitrophenol

has an ultra-violet spectrum of an unusually simple nature, consisting of two bands, one of which

is enhanced in alkaline solution (λmax=400nm) and the other in acid solution (λmax=320nm).

Figure 4. λmax for p-nitrophenol in the acid and base forms.

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C- Calculations, dilutions and solution preparation ..

Dilution Factor

It is an expression which describes the ratio of the aliquot volume to the final volume. For

example, a 1:5 dilution, with a 1:5 dilution factor, (said: "1 to 5" dilution) describes combining 1

unit volume of solute (the material to be diluted, here termed initial volume) with (approximately)

4 unit volumes of the solvent to give 5 units of total final volume. Note that some solutions have

in total slightly less volume than their components. As such, one has to start with the solute

volume and add solvent to bring the mixture to a final indicated volume.

Based on the dilution formula:

MinitialVinitial=MfinalVfinal (a1)
𝑉𝑖𝑛𝑖𝑡𝑖𝑎𝑙
Note that the dilution factor described above = .
𝑉𝑓𝑖𝑛𝑎𝑙

By rearranging the equation (a1), we find that:

𝑽𝒊𝒏𝒊𝒕𝒊𝒂𝒍 𝑴𝒇𝒊𝒏𝒂𝒍
𝒅𝒊𝒍𝒖𝒕𝒊𝒐𝒏 𝒇𝒂𝒄𝒕𝒐𝒓 = = . (a2)
𝑽𝒇𝒊𝒏𝒂𝒍 𝑴𝒊𝒏𝒊𝒕𝒊𝒂𝒍

Note that in Beer Lambert’s law, M is termed c (concentration).

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Serial Dilution

A serial dilution is

the stepwise dilution

of a substance in

solution. Usually the

dilution factor at

each step is constant.

In this experiment, you will learn how to prepare solutions using dilutions, and learn how to use a

spectrophotometer.

Materials
Pipettes
Pipet tips
Water
2.0 M CuSO4 solution
10mM CuSO4 solution
Cuvettes
CuSO4 solution with an unknown concentration

1. Prepare a set of serial dilutions of the 2.0M CuSO4 dilutions: 1000mM, 100mM, 10mM, and

1mM. Measure the absorbance value for each.

2. From the 1000mM you prepared in ‘2’, prepare the following set of serial dilutions: 500mM,

250mM, 125mM. Measure the absorbance value for each.

3. From the 100mM you prepared in ‘2’, prepare the following set of serial dilutions: 50mM,

25mM.

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4. Measurements for the unknown: Prepare 1 ml of the following dilution factors of the unknown

CuSO4 solution using water: 1:2, 1:5, 1:10, 1:50, and 1:100.

5. Set the spectrophotometer to wavelength 700 nm and blank against water. Read the

Absorbance at 700nm of all the solutions prepared.

6. Plot all the A vs c values to one graph. On the graph, find the unknown values. Calculate the

average and standard error for the unknown readings you get.

Questions
1. Determine the A700nm for each dilution.
2. Determine the concentration of the unknown CuSO4 solution.
3. Why is a solution of CuSO 4 blue?

15
 1D. Preparation of buffer solutions

Living things are very sensitive to changes in the concentrations of hydrogen ions (H+)
and hydroxide ions (OH-). Homeostasis is the maintenance of a constant internal
environment in terms of temperature, pH, and water concentrations, etc.

The internal pH of most living cells is close to 7. Even slight change in pH can be harmful,
because all chemical reactions of the cell are very sensitive to H+ and hydroxide ions (OH-)
concentrations.

Remember that:
1. In pure water, the concentrations of these ions are equal.
2. The pH scale is used to express the concentration of hydrogen ions in a solution. pH is
defined as the negative logarithm of the hydrogen ion concentration.
3. In a neutral solution like pure water, the hydrogen ion concentration is 10 -7 molar, so the pH
of a neutral solution is pH 7-- the midpoint of the pH scale.
4. In a neutral solution, the H+ and OH- concentrations are equal.
5. An acid is any substance that increases the H+ concentration of a solution. A base is any
substance that reduces the H+ concentration of a solution.

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Biochemical Buffers:

Buffers ions are used to maintain solutions at constant pH values.

Weak acids and bases do not completely dissociate in solution but exist as equilibrium mixtures.
k2
 HA <--> H+ + A-
k1

HA represents a weak acid

A- represents the conjugate base
k1 represents the rate constant for dissociation of the acid
k2 represents the associate rate of the conjugate base and the hydrogen ion.

The equilibrium constant Ka, for the weak acid HA is defined as:

 Ka = k1/k2 = [H+][ A-]/ [HA]

Which can be rearranged to define [H+]:

 [H+] = Ka[A-]/ [HA]

[H+] is often reported as pH, which is -log[H+].

In a similar fashion. -log Ka is represented by pKa , which is -log Ka.

By substituting [H+] and Ka by pH and pKa, the [H+] = Ka[ A-]/ [HA] can be converted to:

 pH = pKa + log [A-]/ [HA]

17
This equation is known as the Henderson-Hasselbalch equation. This equation defines the
relationship between pH and the ratio of acid and conjugate base concentrations.
The Henderson-Hasselbalch equation is of great value in buffer chemistry because it can be used
to calculate the pH of a solution if the molar ratio of buffer ions ([A -]/ [HA]) and the pKa of HA
are known. Also, the molar ratio of HA to A- that is necessary to prepare a buffer solution at a
specific pH can be calculated if the pKa is known.
A solution containing both AH and A- has the capacity to resist changes in pH, it acts as a buffer.
If acid (H+) were added to the solution, it would be neutralized by A- in solution:

 H+ + A-  HA

Base added to the buffer solution would be neutralized by reaction with HA:

 OH- + HA  A- + H2O

The most effective buffering systems contains equal concentrations of the acid and the conjugate
base. According to the Henderson-Hasselbalch equation, when [A-] is equal to [HA] pH equals
pKa. Therefore, the pKa of a weak acid-base system represents the center of the buffering region.
The effective range of a buffer system is generally two pH units, centered at the pKa value.

 Effective range for a buffer = pKa ± 1

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Blood buffer systems, pH 7.4:
The main buffer system in human blood is the CO2/bicarbonate system:

CO2 + H2O <--> H2CO3 <--> H+ + HCO3-

The pKa of this system is 6.1. This buffer system is an open system, because CO 2 is gas and can
be easily added or eliminated from the system.
Other buffer systems are the phosphate buffer, the plasma proteins, and the hemoglobin (in
erythrocytes).

19
Questions:
1A. Calculate the pH of the following buffer:
10 ml of 0.1M CH3COOH + 5 ml 0.1 N NaOH, the pKa of the acetic acid is 4.7.
1B. Prepare the above mentioned solution and measure its pH.

2A. Calculate the pH of the following buffer:

10 ml of 0.1M CH3COOH + 0.2 ml 1 N NaOH, the pKa of the acetic acid is 4.7.
2B. Prepare the above mentioned solution and measure its pH.

3A. Calculate the pH of the following buffer:

10 ml 0.15M Na2HPO4 + 2 ml 0.15M NaH2PO4, the pKa is 6.7.

4. Calculate the amounts of 0.3M Na2HPO4 and of 0.3M NaH2PO4 needed to prepare a 0.15 M
phosphate buffer with a pH of 7.4, the pKa is 6.7.

5. For the preparation of buffer solution you have a 0.1M acetic acid pKa = 4.7 and 0.1M sodium
acetate.
a. Add 5 ml of 0.1M sodium acetate to 15 ml of 0.1M acetic acid. Calculate the expected pH of
the solution according to Henderson-Hasselbalch equation.
b. Measure the pH value of the prepared solution and compare the calculate value with the
measured value.
d. Prepare 20 ml of 0.1M acetate buffer pH 5.0.
6. Determine the pKa value of acetic acid:
a. To 10 ml of 0.1 M acetic acid add step wise (1 ml each time) 15 ml of 0.1 M NaOH and then
measure the pH of the solution (after the addition of each ml of NaOH).
b. B. Draw the titration curve (pH angainst the amount of NaOH) of the solution.
c. Determine the pKa of the acetic acid.
pH = pKa when [A-] = [HA]

20
'

Laboratory 2

2A. Titration curves of amino acids

Objective:
To Determine the identity of unknown amino acids through their PKa values.

Introduction:

- Twenty different amino acids are used to build proteins. The various amino acids can
be linked in almost any sequence, to form an almost infinite variety of different
proteins.
- Amino acids are organic molecules having a carboxyl group, an amino group, an
hydrogen linked to the central c-atom.

- Nine of the twenty amino acids are essential.

H
- The structure of amino acids is: 2HN-C-COOH
R

- The main backbone of every amino acid is the same (3HN-CH-COOH). The R-group,
or side chain (which projects out from the backbone) makes each of the twenty kinds
of amino acids unique.
- The backbone of amino acids contain two ionizable groups:
1. the -carboxyl group
2. the -amino group.
- Amino acids in solution at neutral pH are predominantly dipolar ions (zwitterions):
The amino group is protonated (-NH3+) and the carboxyl group is dissociated (-COO-).
- The ionization state of an amino acid varies with pH:

1. at pH 1: 2HN-C-COOH  3HN+-C-COOH

2. at pH 7: 2HN-C-COOH  3HN+-C-COO-

21
 3. at pH 11 : 2HN-C-COOH  2HN-C-COO-

Acidic and basic amino acids contain an ionizable side chain.

The iso-electric point (IEP):

The IEP is the pH value at which the net charge of the amino acid is 0. Each amino acid has its

specific IEP.

The pKa of the carboxyl group is called pK1, and the pKa of the amino group is called pK2.

At the IEP:

pH = 1/2 (pK1 + pK2)

For example, Alanine:

pK1 = 2.3, pK2 = 9.9, IEP = (2.3 + 9.9)/2 = 6.1

So if the side chain does not have an ionizable group, then the pI is simply the average

of the α- NH3 and α-COOH pKa values.

If the side chain has an ionizable group then all three pKa values must be considered. •

If the side chain is acidic (asp and glu), then average the sidechain pKa with the α-COOH

pKa.

• If the side chain is basic (his, arg, and lys), then average the sidechain pKa with the α-

NH3 pKa. •

For other ionizable groups (tyr and cys), determine which is the middle pKa and average

it with the α-COOH pKa..

22
 Titration curve:

Titration curves are produced by monitoring the PH of agiven volume of asample solution after

successive addition of acid or alkaline.so it represent a plot of PH against the volume of titrant

added or against the number of equivelents added per mole of the sample. Further more the curve

will have two breaks for simple amino acid(diprotic) in PH because because more than one acidic

portion will be given off.

and this ionization follow Henderson–Hasselbalch equation

PH=PKa +log unprotonated form  protonated.

pH = pKa + log ([A-]/[HA])

According to this equation, PKa defined as the PH at which the concentration of protonated and

un protonated form of a particular ionizable species are equal. Also equal the PH at which the

ionizable group is at its best buffering capacity.

Fig.Titration curve of Glycine

23
Table 1: The pK values of some amino acids:


Amino acid -carboxyl group -amino group Side chain

Alanine 2.3 9.9

Glycine 2.4 9.8

Phenylalanine 1.8 9.1

Serine 2.1 9.2

Valine 2.3 9.6

Glutamic acid 2.2 9.7 4.3

Histidine 1.8 9.2 6.0

Cysteine 1.8 10.8 8.3

Tyrosine 2.2 9.1 10.9

Lysine 2.2 9.2 10.8

Arginine 1.8 9.0 12.5

24
Procedure:

Identifying of unknown amino acids by measuring their pK a values

1. Standardize your pH meter against pH 4 and 7

2. Place 50 ml of the amino acid solution A in a beaker

3. Wash the electrode with dest water

4. Immerse the electrode in the solution.

5. Put 50 - 100 ml 0.15 M NaOH in a new beaker

6. Titrate the amino acid solution by adding 1 ml aliquots of 0.15 M NaOH, mix

well and measure the pH. (Be very careful not to damage the electrode).

7. Continue titrating until reaching pH 12.5-13

8. Repeat the above procedure (steps 1-7) with amino acid solution B.

9. Prepare the titration curves of solution A and B by plotting the the pH values vs.

ml of NaOH.

10. Determine the pKa values of the amino acids and identify the amino acids with the

help of Table 1.

* Amino Acids (A.A) are prepared by technicians as follows:

Solution = 1.5ml HCl (32%) is added to 1 liter DDW , pH~1.5

10mM of A.A dissolved in the above solution.

25
 2B. Thin Layer Chromatography (TLC)

Introduction
Planar chromatograpy belongs to the family of chromatographic methods. It is a method for
separation and determination of substances, allowing to carry out qualitative and quantitative
analysis of chemical components in complex mixtures. Thin-layer chromatography (TLC) is a
chromatographic technique that is useful for separating organic compounds. Because of the
simplicity and rapidity of TLC, it is often used to monitor the progress of organic reactions and to
check the purity of products.

A TLC plate is a sheet of glass, metal, or plastic which is coated

with a thin layer of a solid adsorbent (usually silica or alumina). A
small amount of the mixture to be analyzed is spotted near the
bottom of this plate. The TLC plate is then placed in a shallow
pool of a solvent in a developing chamber so that only the very
bottom of the plate is in the liquid. This liquid, or the eluent, is the mobile phase, and it slowly
rises up the TLC plate by capillary action.

As the solvent moves past the spot that was applied, an equilibrium is
established for each component of the mixture between the molecules of
that component which are adsorbed on the solid and the molecules which
are in solution. In principle, the components will differ in solubility and in
the strength of their adsorption to the adsorbent and some components will be carried farther up
the plate than others. When the solvent has reached the top of the plate, the plate is removed from
the developing chamber, dried, and the separated components of the mixture are visualized. If the
compounds are colored, visualization is straightforward. Usually the compounds are not colored,
so a dye is applied or UV lamp is used to visualize the plates. (The plate itself contains a
fluorescent dye which glows everywhere except where an organic compound is on the plate.)

26
 TLC Solvents Choice

When you need to determine the best solvent or mixture of solvents (a "solvent system") to
develop a TLC plate or chromatography column loaded with an unknown mixture, vary the
polarity of the solvent in several trial runs: a process of trial and error. Carefully observe and
record the results of the chromatography in each solvent system. You will find that as you
increase the polarity of the solvent system, all the components of the mixture move faster (and
vice versa with lowering the polarity). The ideal solvent system is simply the system that gives
the best separation.

The Rf value

The retention factor, or Rf, is defined as the distance traveled by the compound divided by the
distance traveled by the solvent.

For example, if a compound travels 2.1 cm and the solvent front travels 2.8 cm, the R f is 0.75:

The Rf for a compound is a constant from one experiment to the next only if the chromatography
conditions below are also constant:

 solvent system
 adsorbent
 thickness of the adsorbent
 amount of material spotted
 temperature

27
Since these factors are difficult to keep constant from experiment to experiment, relative R f
values are generally considered. "Relative R f" means that the values are reported relative to a
standard, or it means that you compare the Rf values of compounds run on the same plate at the
same time.

The larger an Rf of a compound, the larger the distance it travels on the TLC plate. When
comparing two different compounds run under identical chromatography conditions, the
compound with the larger Rf is less polar because it interacts less strongly with the polar
adsorbent on the TLC plate. Conversely, if you know the structures of the compounds in a
mixture, you can predict that a compound of low polarity will have a larger R f value than a polar
compound run on the same plate.

Amino acids
Amino acids are the building blocks of peptides and proteins. They possess two functional
groups— the carboxylic acid group gives the acidic character, and the amino group provides the
basic character. The common structure of all amino acids is:

The R represents the side chain that is different for each of the amino acids that are commonly
found in proteins. However, all 20 amino acids have a free carboxylic acid group and a free
amino (primary amine) group, except proline which has a cyclic side chain and a secondary
amino group. The properties provided by these groups are used to characterize the amino acids.

28
The common carboxylic acid and amino groups provide the acid-base nature of the amino acids.
The different side chains, and the solubilities provided by these side chains, affect their rate of
migration in thin-layer chromatography (TLC).

Ninhydrin
Ninhydrin (2,2-Dihydroxyindane-1,3-dione) is a white solid chemical which is soluble in ethanol
and acetone at room temperature. It is used to detect amines and ammonia. When reacting with
these free amines, a deep blue or purple color known is produced. Ninhydrin is most commonly
used to detect fingerprints, as the terminal amines of lysine residues in peptides and proteins
sloughed off in fingerprints react with ninhydrin. You may try it in this lab. We will use
ninhydrin in our lab to detect amino acids at the TLC plate. Ninhydrin can also be used to
monitor peptide synthesis.
When ninhydrin reacts with amino acids, the reaction releases CO 2. The carbon in this CO2
originates from the carboxyl carbon of the amino acid. This reaction has been used to release the
carboxyl carbons of bone collagen from ancient bones. for stable isotope analysis.

Aspartame

29
Aspartame an artificial sweetener, is approximately 200 times sweeter than sucrose, or table
sugar used as a sugar substitute in some foods and beverages. In Aspartame is a methyl ester of
the aspartic acid/phenylalanine dipeptide. It was first synthesized in 1965 and sold under the
brand name NutraSweet; since 2009 it also has been sold under the brand name AminoSweet.
However, because its breakdown products include phenylalanine, aspartame must be avoided by
people with the genetic condition phenylketonuria (PKU).
Aspartame is the methyl ester of the dipeptide aspartylphenylalanine. Upon hydrolysis with HCl
it yields aspartic acid, phenylalanine, and methyl alcohol. When this artificial sweetener was
approved by the Food and Drug Administration, opponents of aspartame claimed that it is a
health hazard, because aspartame would be hydrolyzed and would yield poisonous methyl
alcohol in soft drinks that are stored over long periods of time. The Food and Drug
Administration ruled, however, that aspartame is sufficiently stable and fit for human
consumption. Only a warning must be put on the labels of foods containing aspartame. This
warning is for patients suffering from phenylketonurea who cannot tolerate phenylalanine.

Aspartame

30
Phenylketonuria
Phenylalanine is an amino acid. A genetic disorder in which the body is not able to break down or
use phenylalanine is called phenylketonuria or PKU. If an individual with PKU consumes too
much phenylalanine he or she can suffer from brain damage that may cause mental retardation,
seizures, and growth delays. Phenylalanine is found in beef, milk, eggs, and artificial sweeteners,
so individuals with PKU have to be very careful about what they eat. Any product containing
phenylalanine must have a warning to help those with PKU avoid those products.

In the present experiment the Rf values of three amino acids, phenylalanine, aspartic acid, and
leucine, in addition to the artificial sweetener aspartame, will be determined. Aspartame will also
be hydrolyzed using HCl as a catalyst to see if the hydrolysis products prove that the sweetener is
truly aspartame. Finally, the analysis of Diet Coke will indicate if the aspartame was hydrolyzed
at all during the processing and storing of the soft drink.

EQUIPMENT/MATERIALS
gloves, foil, ruler, pipets, capillary tubes, test tubes and rack, heat gun
chromatography chambers
TLC silica gel plates
butanol: acetic acid: water-solvent
1% solutions of aspartic acid, phenylalanine, and leucine
1-2 gr Aspartame
3 M HCl
2% ninhydrin

31
Procedure
1. Tare a weigh boat on a top-loading balance. Mass between 0.1 and 0.2 g of Aspartame. in
a small test tube. Similarly, prepare another test tube.
2. Add 1 ml of 3 M HCl to one the test tubes and boil it for 30 seconds. Add 1 ml of DDW
to the next test tube.
3. Label 4 small test tubes, respectively, for aspartic acid, phenylalanine, leucine, and Diet
Coke. Using the labeled pipets, transfer about 0.5 ml of each solution into the matching
test tube.
4. Add enough solvent to a depth of approximately 1 cm or less in your developing tank.
5. Get your silica gel plate. Carefully hold the plate by the sides to prevent disturbing the
silica gel layer. Draw a pencil line about 1.5 cm from the bottom plate.
6. Mark one point on the line for each one of your known and unknown solutions. 1 cm on
both sides. Number each point.
7. At point number 1 apply a very small drop of one of your known solutions. Do not wet
the silica beyond a diameter of 2-3 mm. Locating the center of large spots will be difficult
later when the spot has moved along the paper.
8. After the liquid has evaporated (only a few seconds), add a second drop to the same spot.
Record the name of the amino acid and the number of the spot.
9. Repeat this procedure for the remaining solution. Remember to record the name of the
amino acid or unknown number and the number of the spot.
10. Allow all the spots to dry completely.
11. Place your TLC plate in the developing tank with the mobile phase with the spots toward
the bottom
12. Allow the solvent to ascend the silica gel to at least ¾ of its height, which will to require 1
hour or less. (The farther the solvent ascends, the greater the separation. Immediately
remove the plate, if the solvent reaches the top.).
13. Remove the plate and quickly mark the farthest advance of solvent front with a pencil,
unless it reached the top of the paper.
14. Dry the plate with a heat gun. Be careful to move the heat gun around and not heat one
point continuously. Do this procedure in the hood.
15. Spray the plate with the 2% ninhydrin solution in the hood.

32
 16. Do not allow the ninhydrin solution to stream down the plate, because this may move
some of the compounds.
17. Dry the plate again with the heat gun. Do not over heat the plate. Long heating times may
cause browning of the plate over the entire surface.
18. Circle each colored spot with a pencil. The ninhydrin spots fade gradually, so circle at
once.
19. Measure the distance from the origin to the center of each colored spot and calculate the
Rf values for all spots.
20. Record the Rf values and the color of each ninhydrin spot.
21. Identify the unknown amino acids.

Questions
1. Why does touching the silica gel with your hands potentially contaminate your plate?
2. Why can an Rf value never be greater than 1?
3. What would happen if so much solvent was used (mobile phase) that the original spots
were covered with solvent?
4. What would happen if you made the line and points with an ink pen rather than a pencil?

33
 2C. Analysis of Biomolecules
Introduction
- Living organisms consist of two main classes of compounds: inorganic compounds and
organic compounds.
- Inorganic compounds include water and minerals that are associated with the organic
compounds of living organisms.
- Organic compounds contain carbon atoms covalently bonded to oxygen, hydrogen, nitrogen,
sulfur or phosphorus. Four classes of macromolecules are essential for life: Proteins, fats,
nucleic acids, and carbohydrates
- Most of the macromolecules are large polymers: polymers are large molecules consisting of
many buildings units (monomers) linked by covalent bonds.
- The main monomers are:
- Amino acids: monomers of proteins
- Monosaccharides: monomers of carbohydrates
- Nucleotides: monomers of nucleic acids

Synthesis of macromolecules: addition of monomers to the polymer chain by condensation or

dehydration reaction. Dehydration reaction is an anabolic process by which two molecules are
chemically bonded through the use of enzymes and a loss of water. Example: glucose + glucose =
maltose + water.
Degradation of macromolecules by hydrolysis. Hydrolysis is a catabolic process by which the
bonds between monomers are broken by the enzyme and the addition of water. Example: Sucrose
+ water = glucose + fructose.

34
Carbohydrates: Carbohydrates include sugars and polymers of sugars. These molecules are used
as building materials and sources of energy. Carbohydrates can be divided into three major
classes:
Monosaccharides: are single sugar molecules, are important as building blocks and cellular
fuels. For example Glucose
Disaccharides: are two molecules of monosaccharides, are important as building blocks and
cellular fuels. For example sucrose
Polysaccharides: are long chains of sugars. For example, cellulose and starch in plant cells and
glycogen in animal cells. Polysaccharides on cell membranes act as cell identification tags.

Lipids: Are a group of polymers that have one characteristic in common, they do not mix with
water. They are hydrophobic. Lipids are stored in cells in form of droplets containing fats. Fat
cells in animals are almost completely filled with fat. The main important groups are: fats,
phospholipids, and steroids.

A. Fats:
- fats are large molecules composed of 2 types of monomers, glycerol (an alcohol containing 3
carbons)and 3 fatty acid molecules.
- The fatty acids in a fat can vary in length.
- The bond connecting the glycerol and fatty acids in the fat molecule is called an ester bond.
- There are two types of fatty acids: saturated and unsaturated.

Saturated Unsaturated
solid at room temperature liquid at room temperature
found mostly in animals found mostly in plants
no double bonds between carbons double bonds found between carbons

35
B. Phospholipids: Phospholipids are important components of cell membranes. Like fats,
phospholipid molecules contain fatty acid tails linked to a glycerol, but in phospholipids a group
containing phosphate replaces one of the tails. This makes the molecule amphipathic; they exhibit
a polar and nonpolar quality. The phosphate group is hydrophilic while the fatty acid area is
hydrophobic.

C. Streroids: All steroids have the same carbon skeleton made of four linked rings; they differ in
what is attached to the rings.

Proteins: Proteins are the most complicated molecules known. A cell contains thousands of
kinds of proteins, which carry out a variety of functions. They make up 50% of the dry weight of
most cells. In most cases, a protein's function depends on its complex three-dimensional
structure.

Protein Structure:
- Proteins are polymers of amino acids.
- Amino acids are linked by peptide bonds, an amino acid polymer of 2-10 monomers is called
a peptide and polymers longer than 10 amino acids are often called a protein or polypeptide.
- Most proteins are folded into a complex globular shape, or conformation. Each protein
molecule consists of one or more chains of amino acid monomers.
- Proteins have four levels of structure: Primary, secondary, tertiary, quaternary.

Nucleic acids
- Nucleic acids are the cell's information molecules.
- One information unit is called a gene and it stores the information for the synthesis of one
polypeptide.
- There are two kinds of nucleic acids: Deoxyribonucleic acid (DNA), and ribonucleic acid
(RNA).
- Nucleic acids are polymers; the monomers are called nucleotides (4 different molecules).
- Each of the four nucleotides consists of a pentose (5) sugar, a phosphate group, and a
nitrogen-containing base

36
In this laboratory you will study organic molecules. Various chemicals will be used to test for the
presence of these molecules. Most often, the result of the test is based on the presence or absence
of a color change. If a color change is observed, the test is positive, indicating that a particular
molecule is present. If color change is not observed, the test is negative, and the molecule is not
present.

1. Detection of Carbohydrates
A. Benedict Test : Carbohydrates are classified as reducing and nonreducing sugars. The
reducing sugars are those which contain free aldehyde or free ketone groups. Monosaccharides
and some disaccharides have these free groups and are reducing sugars. Benedict’s reagent
(copper sulfate in sodium hydroxide solution) oxidizes aldehyde or ketone groups by reducing
copper from Cu++ to Cu+ forming a red precipitate, Cu2O. Besides the presence of sugar, the test
shows the amount of reducing sugar present in the solution. A reaction with a small amount of
reducing sugar turns the reagent green. A sample with a large amount of reducing sugar gives
red-orange color.

B. Lugol's Test (Iodine Test): The test is used to distinguish starch from Mono-, di-, and other
polysaccharides. Starch is a polymer of glucose, which is coiled up in a particular way so it can
interact with iodine molecules in Lugol's solution to give a blue-black color. Other polymers even
those of glucose, lack the precise coiled structure of starch and do not give the blue color. A
violet-brown to red-brown color is given by cellulose, and a red color is given by glycogen.

37
2. Detection of lipids
Sudan III Dye Test: This test uses a dye that reacts with long chains of carbon atoms in the
fatty acid part of the lipid molecule. These long fatty acids make lipids nonpolar and therefore
immiscible in water. Sudan III dye dissolves in nonpolar substances and stains them red.

3. Detection of proteins
A. Ninhydrin Test: Ninhydrin is a reagent that reacts with free amino groups (-NH2) of an
amino acid or a protein. It turns violet when applied to a amino acid or protein-containing
solution, except with proline, it gives a yellow color.

B. Biuret Test: Biuret reagent (blue color) contains a strong solution of sodium or potassium
hydroxide (NaOH or KOH) and a very small amount of very dilute copper sulfate (CuSO 4)
solution. The reagent changes color in the presence of a protein and peptides, because the
amino group complexes with the copper ions.
A violet color is indicative of whole proteins, whereas pink or rose color is indicative of
polypeptides.

Procedure

In the experimental procedures you perform, you will need to include a distilled water

sample, which is known as a control. The control sample will go through all the steps of the

experiment and give a negative test result, so you will be able to distinguish the difference

between a positive and a negative result.

38
1. Detection of Carbohydrates

A. Benedict Test

1. Label four clean tubes (a-d). Place 1ml of the following solutions in the
corresponding tubes:
a. 2% glucose.
b. 2% starch.
c. 2% sucrose.
d. DDW.
2% glucose
b. 2% starch
c. 2% succrose
d. distilled water.

2. Add 4ml of benedict’s solution to each tube.

3. Place the tubes in a boiling water bath for 2 minutes.
4. Note the formation of precipitate and any color change.
5. Record your results and explain your results

39
Hydrolysis of polysaccharides

Polysaccharides contain only one reducing group for several hundred residues so that they are

effectively nonreducing. Acid hydrolysis gives the constituent monosaccharides.

1. Label 4 clean tubes (a-d). Place 3ml of the starch solution ( 10 g/ml) in each tube

2. Add 3 drops of 11M HCl to each tube

3. Boil tube (b) for two min. tube (c) for 4 min, and tube (d) for 6 min, keep tube (a) without

boiling.

4. Place one ml of a-d in a new labeled tube (a1-d1) and neutralize it with 5.5M NaOH. Bring

the solution to pH~7, check with pH paper.

5. Prepare additional 4 control tubes: 2% starch, 2% glucose, 2% glucose with HCl, 2% glucose

with NaOH (why do you need these controls?).

6. Add 4ml of benedict’s solution

7. Place the tubes in a boiling water bath for 2 minutes.

8. Note the formation of precipitate and any color change.

9. Record your results and explain your results

40
2. Detection of lipids

Sudan III Dye Test

1. Label 4 test tubes (a-d).

2. Place the following solutions in the corresponding tubes:

a. 1 ml oil + 2ml water

b. 3 ml 2% starch

c. 3 ml milk

d. 3 ml distilled water

3. Add a two drops of Sudan III, shake well and wait 2 minutes.

4. Record and explain your results

41
Detection of proteins

A. Ninhydrin Test

Caution: Ninhydrin is a toxic substance, avoid inhaling the fumes, and do not let the

solution touch your skin or clothing.

1. Label three test tubes (a-c).

2. Place 1ml of the following solutions in the corresponding test tubes.

a. 2% Glycin

b. 2% Bovine serum albumin

c. milk

d. distilled water

3. Add 1ml of ninhydrin solution to each test tube.

4. Place the test tubes in boiling water for 10 minutes.

5. Remove the test tubes and place them in a beaker containing tap water.

6. Record and explain your results.

42
 Laboratory 3

3A. Isolation and purification of proteins

Introduction:
Proteins can be separated from each other on the basis of their physical and chemical properties,
including, water solubility, size, and charge.
Proteins can be purified according to charge (either by electrophoresis or ion exchange
chromatography), size differences (by Dialysis or gel filtration), solubility differences (by
precipitation), or binding affinity.

1. Isolation of casein from milk

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 phosphorus containing proteins and not a single compound.
Most proteins show a minimum solubility at their isoelectric point (IEP). The IEP is the pH value
at which the net charge of the protein is 0. This principle is used to isolate the casein by adjusting
the pH of the milk to pH 4.8 (the IEP of casein). Casein is also insoluble in ethanol and this
property is used to remove unwanted fat from the preparation.

Method:

1. Calibrate the pH meter.

2. Place 100 ml of milk in a 500 ml beaker add 100 ml of tap water and warm to 40 oC (control

the temperature with Thermometer).

3. Slowly with stirring (use magnet stirrer) add about 1 ml of 5% HCl.

4. Keep adding HCl (drop wise) until the pH of the solution lowered to pH 4.8 (use pH meter).

5. Stir for 10 min, then allow the solution to settle for further 10 min.

6. Decant the supernatant (after centrifugation 3000Xg, 5min).

7. Wash the precipitate three times with small volume of distilled water, (centrifuge for 2 min.

each time).

43
8. Collect the precipitate by centrifugation.

9. Wash the precipitate with 95% ethanol and centrifuge for 2 min.

10. Wash the precipitate with 10 ml acetone and air-dry it.

11. Weight the casein and calculate the percentage yield of the protein and compare with 3.5 g

theoretical yield from 100 ml milk.

2. Isolation of albumin by precipitation:

The solibility of most proteins is lowered at high salt concentrations. This effect, called salting
out, is very useful. The dependence of solubility on salt concentration differs from one protein to
another. For Example, 0.8 M ammonium sulfate precipitates fibrinogen, whereas 2.4 M is needed
to precipitate serum albumin.

Method:

1. Crack an egg and separate the yolk

2. Add the white to 100 ml distilled water in a beaker.

3. Stir the mixture slowly until the globulins precipitate out

4. Centrifuge the suspension at 3000 g for 10 min

5. To the supernatant (albumin) add solid ammonium sulfate (40 g/100ml) with slow sirring

until the albumin precipitate out.

6. Centrifuge the suspension at 3000 g for 10 min.

7. Collect the precipitate, place it on a watch glass air dry it

8. Weight the albumin and determine its concentration per an egg

For the Technician:

44
Buiret reagent preparation:

For 200ml add the following reagents:

1. 6 gr NaOH

2. 1.2 gr Na/K tartarate

Dissolve.

3. 0.3 gr CuSO4

45
 3B. FRACTIONATION OF PLASMA/SERUM PROTEINS

The solubility of most proteins is lowered at high salt concentrations. This effect, called salting
out, is very useful. The dependence of solubility on salt concentration differs from one protein to
another. For Example, 0.8 M ammonium sulfate precipitates fibrinogen, whereas 2.4 M is needed
to precipitate serum albumin.

Method:
1. Place 5 ml of plasma in a 10-ml centrifuge tube.

2. (Start here if you got plasma fraction)Slowly add solid ammonium sulfate to bring the

mixture to 25% saturation (ammonium sulfate max solubility is 74.4 g/100 mL at 20

°C).

3. Mix the mixture well and leave it on your desk for 5 min.

4. Centrifuge the mixture at 3000 rpm for 10 min. The precipitate contains fibrinogen (if

you got serum fraction).

5. (Start here if you got serum fraction) Pipet the supernatant into a new 10-ml

centrifuge tube and bring it 46% ammonium sulfate (from 25 to 46%).

6. Mix the mixture well and leave it on your desk for 5 min.

7. Centrifuge the mixture at 3000 rpm for 10 min. The precipitate contains globulins.

8. Pipette the supernatant into a new 10-ml centrifuge tube and bring it to 64% saturation

(from 46 to 64%).

9. Mix the mixture well and leave it on your desk for 5 min.

10. Zentrifuge the mixture at 3000 rpm for 10 min. The precipitate contains albumin.

11. Decant the supernatant.

12. Suspend each of the three precipitates by dissolving the precipitate in 3 ml water plus

one or more drops of 10 M NaOH.

46
Protein dretermination:

1. Add 0.8 ml of Biuret Reagent to 0.2 ml of each protein suspension

2. Mix well

3. Incubate all tubes for 5 min at room temperature

4. Measure the absorbance at 546 nm of 0.2 ml of the mixture in an ELISA reader plate.

5. Using the measured values, determine the relative protein concentration of the isolated

proteins

47
 3C. Photometric determination of protein concentration

Introduction:

Spectrophotometers: see the introduction of lab 1C.

Quantitative methods for protein concentration Measurement:

A wide variety of photometric methods are available for the determination of protein

concentrations. The commonly used methods are:

1. Biuret: In alkaline solution Cu2+ complexes with peptide bonds of polypeptides giving a

purple color. The color intensity is proportional to the number of peptide bonds and therefore, to

the polypeptide concentration. The determination limit of this assay is about 250 g/ml. The

absorption peak of Cu2+-polypeptide complex is at 546 nm.

2. Absorption at 280 nm (A280): this direct method is based on the fact that two amino acids

(tyrosine and tryptophan) have a maximum absorbance at 280 nm. Since the amount of tyrosine

and tryptophan differ from one protein to another, this method is suitable only for pure proteins

with an known extinction coefficient.

3. BCA: The BCA protein assay is a highly sensitive method for the spectrophotometric

determination of protein concentration. This method combine Biuret Cu 2+-polypeptide complex

with a highly sensitive detection reagent for the Cu2+-polypeptide complex. The absorption

maximum of Cu2+-polypeptide complex is at 562 nm. The detection limit of the BCA is about 20

g/ml.

48
4. Bradford: Like BCA this method is highly sensitive. The dye used in this assay is Coomassie

blue. The absorbance maximum is at 595 nm. The detection limit is about 5g/ml. 

Procedure:

For the determination of the protein concentration of given samples you have the following

solutions:

A. A standard solution with protein concentration of 5 mg BSA (Bovine serum

albumin)/ml.

B. Samples A and B with unknown protein concentrations.

C. Protein samples purified in lab 3A and 3B.

1. Prepare a set of protein standards of known concentration by diluting the BSA standard

solution (Total volume including the Buiret reagent is 1.25 ml):

a. Label 5 clean tubes (1 -5).

b. Add the following dilutions:

Tube number: 1 2 3 4 5

BSA standard: 0 50 100 150 250 ul

Water: 250 ? ? ? ?

2. Prepare your unknown samples A and B as well as the purified protein from lab # 3A & 3B:

a. Label 6 clean tubes (A1, A2 A3; B1, B2, B3, C1, C2, C3).

b. Add the following dilutions

49
Tube number: A1 A2 A3 B1 B2 B3 C1 C2 C3

Unknown sample A: 50 100 250 0 0 0 0 0 0 ul

Unknown sample B: 0 0 0 50 100 250 0 0 0 ul

Serum 0 0 0 0 0 0 0 10 50ul

Water: 200 ? ? ? ? ? ? ? ? ul

(Similarly prepare 3 tubes for each sample you had purified in lab 3A & 3B).

3. Add 1 ml of Biuret Reagent to each tube (1 -5 and A1, A2 A3; B1, B2, B3 and the tubes

prepared for the purefied protein samples).

(NOTE: it is recommended that you prepare a table for all the samples, that includes all the

reagents and steps).

4. Mix well

5. Incubate all tubes for 5 min at room temperature

6. Measure the absorbance at 546 nm of 0.2 ml of the mixture in ELISA reader.

7. The absorbance in tube number 1 represents the background and should be subtracted from

the absorbance of all other test tubes.

8. Prepare a standard curve by plotting the absorbance at 546 nm vs. protein concentration.

9. Using the standard curve, determine the protein concentration for each unknown sample.

Question:

An ATP solution showed an Absorbance of 0.5 at 260 nm. The molar extinction coefficient
(of ATP is 15 M-1 cm-1. Calculate the concentration of this solution.

50
 3D. PROTEIN ELECTROPHORESIS (SDS-PAGE)

Introduction

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), is a technique used

in biochemistry to separate proteins according to their electrophoretic mobility. The proteins are

first denaturized with some reagents and processes:

1. The proteins are mixed with SDS, an anionic detergent which denatures secondary and non–

disulfide–linked tertiary structures and applies a negative charge to each protein in

proportion to its mass.

2. Dithiothreitol (DTT) or 2- mercaptoethanol (beta-Mercaptoethanol/BME) are added to

reduces the disulfide bonds.

3. A tracking dye is added to the protein solution to allow the experimentor to track the

progress of the protein solution through the gel during the electrophoretic run.

4. Boiling the sample for 3 min, or heating 70C for 5 min.

Take care not to confuse Native PAGE with SDS-PAGE. SDS-PAGE uses SDS (Figure 1) to

denature the proteins and provide a negative charge that is proportional to the protein mass,

allowing electrophoretic separation. Without SDS, different proteins with similar molecular

weights would migrate differently due to differences in folding, as differences in folding patterns

would cause some proteins to better fit through the gel matrix than others. Adding SDS solves

this problem, as it linearizes the proteins so that they may be separated strictly by length (number

of amino acids). The SDS binds to the protein in a ratio of approximately 1.4 g SDS per 1.0 g

protein.

51
The denatured proteins are subsequently applied to one end of a layer of polyacrylamide (Figure

1) gel submerged in a suitable buffer (Figure 2). An electric current is applied across the gel,

causing the negatively charged proteins to migrate across the gel. Depending on their size, each

protein will move differently through the gel matrix: short proteins will more easily fit through

the pores in the gel, while larger ones will have more difficulty

SDS - Sodium dodecyl sulfate

Figure 1

52
Figure 2

Figure 3

It is common to run "marker proteins" of known molecular weight in a separate lane in the gel, in

order to calibrate the gel and determine the weight of unknown proteins by comparing the

distance traveled relative to the marker (Figure 3).

Gel electrophoresis is usually the first choice as an assay of protein purity due to its reliability

and ease. The presence of SDS and the denaturing step causes proteins to be separated solely

based on size. In this experiment you are expected to be familiar with SDS-polyacrylamide gel

electrophoresis (SDS-PAGE) of proteins and determine purity and the molecular weight of the

proteins you had purified.

Materials:

1. 30% Acrylamide/bis (29:1) solution;

53
2. 4 x Resolving gel buffer: 1.5 M Tris-HCl (pH 8.8), 0.4% SDS;

3. 4 x Stacking gel buffer: 0.5 M Tris-HCl (pH 6.8), 0.4% SDS;

4. 1 x Running buffer: 25 mM Tris-HCl (pH 8.3), 250 mM glycine, 0.1% SDS.

5. 3 x protein loading buffer: 0.19 M Tris-HCl (pH 8.0), 3% glycerol, 6% SDS, 300 mM
DTT, and 0.25% bromophhenol blue;

6. 10% Ammonium persulfate (APS);

7. TEMED;

8. Gel staining buffer: 1 g of Coomassie Brilliant Blue dissolved in 400 ml of solution

containing 45% methanol, 45% H2O, and 10% acetic acid;

9. Destaining buffer: 25 % methanol, 10% acetic acid, and 65% H 2O;

10. Gel Drying Buffer: 40% ethanol, 4% glycerol, and 56% H 2O.

11. Protein samples.

12. Protein standards.

54
Experimental Procedure:
Part 1&2 is prepared by the technician.

1. Prepare 40 ml of 12% resolving gel solution as following:

13.68 ml of H2O
16 ml of 30% Acrylamide/bis solution
10 ml of 4 x separating gel buffer
300 μl of 10% APS
20 μl of TEMED
First assemble the gel caster, and add the 15% gel mix to gel caster. Overlay the solution
gently with water or 1-butanol saturated with H2O, and allow to be polymerized. You
should see the gel-H2O interface disappear and then reappear when the gel is polymerized;

2. Prepare 20 ml of 4% stacking gel solution as following:

12.1 ml of H2O
2.7 ml of 30% Acrylamide/bis solution
5 ml of 4 x stacking gel buffer
200 μl of 10% APS
10 μl of TEMED
Add the gel mix to the gel caster, and insert the comb to the gel solution; you should wait
until gel is fully polymerized. Once the gel is polymerized, the comb can be removed
gently. Then assemble the gel into the electrophoresis apparatus.

3. Load your samples:

Mix two volumes of your samples with 1 volume of 3 x sample loading buffer, and heat

the samples over 700C for 5 min, and load about 20 μl of your samples to each well of the gel.

Make sure that you load about 50μg protein to each lane. You may want first to dilute your

sample to 2-5 mg/ml ( = 2-5 μg/ μl).

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To make it easier, it is recommended to dilute all the protein sample to 2.5 μg/ μl, take 20 μl from

each sample to a tube containing 10 μl 3 x sample loading buffer, heat and add load all the

sample to the lane.

Also add the protein standards in one or two lanes on each gel.

4. Run the gels:

Apply power at 70-80 V until the dye has reached the top of the resolving gel, and then

increase the power into 120 V. When the dye reaches the bottom of the resolving gel, turn

off the power supplier. Disassemble the gel apparatus, and stain the gel for about one

hour at Gel staining buffer. Destain your gel at the destaining buffer until the protein

bands are clearly seen in the gel, and dry the gel if it is possible.

5. Determination of molecular weight of the protein provided in your class:

Measure the migration of proteins and the dye, and calculate the relative mobility (Rf) of each

protein by using the following equation.

Rf = (Distance of the protein migrated)/ Distance of the dye migrated)

Plot the mobility versus log value of molecular weight of the stand proteins, and then determine

the molecular weight of the protein sample.

Discuss the protein purity level in the deferent samples.

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 Laboratory 4

Enzyme Action

Introduction

Cells contain many different molecules that can engage in a variety of chemical reactions.
When molecules react, their atoms and bonds are rearranged. Reactants combine to form
products. Reactant and product molecules store free energy in the arrangements of their atoms
and bonds. Reactions involve changes in bonding and changes in free energy.
There are two types of chemical reactions; exergonic reactions and endergonic reactions.

Exergonic reactions are "downhill" changes that release free energy

Endergonic reactions are "uphill" changes; they absorb free energy from their surroundings.

If exergonic reactions occur spontaneously, what keeps molecules from breaking apart
and cell chemistry from racing out of control?
For any reaction to occur even a downhill reaction, some energy must be added to get the
reaction going. This energy is needed to break bonds in the reactant molecules. The energy is
then released when the bonds in the product molecules form. The energy needed to start a
chemical reaction is called the energy of activation (EA). This required energy input
represents a barrier that prevents even energy-releasing exergonic reactions from occurring
without some added energy.

How does a living cell overcome the energy barrier so that its metabolic reactions can
occur quickly and precisely?
Enzymes: An enzyme does not add energy to a reaction; instead, it speeds up a reaction by
lowering the energy of activation. Enzymes are very selective. Its three-dimensional shape
allows it to act only on specific molecules, referred to as the enzyme's substrates. As the
substrates bind to the enzyme's active site, they are held in a position that facilitates the
formation of new chemical bonds. This takes less activation energy than the unaided reaction.
Products form and are released. The enzyme emerges unchanged from the reaction.

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 During the reaction, substrates (S) combine with special regions of the enzyme (E) called
active sites to form a temporary enzyme-substrate complex (ES) and then a enzyme-product
complex (EP):
S + E  ES  EP  E + P
Each enzyme is specific for a certain reaction. The specificity is a result of a unique amino
acid sequence, which causes it to have a unique three-dimensional structure. Any physical or
chemical factor that blocks or changes the shape of the active site in the enzyme will interfere
with the activity and efficiency of the enzyme. If the change is large enough, the enzyme will no
longer function at all, and is said to be denatured. There are several factors that are especially
important in determining the enzyme's shape, and these are regulated both in the living organisms
and in laboratory experiments to give optimum enzyme activity. These factors include
temperature, pH, enzyme concentration and substrate concentration.
Enzyme kinetics is the study of the rates of chemical reactions catalysed by enzymes. The
study of an enzyme's kinetics provides insights into the catalytic mechanism of this enzyme, its
role in metabolism, how its activity is controlled in the cell and how drugs and poisons can
inhibit its activity. In this laboratory, we will determine the Michaelis constant and the maximal
velocity of alkaline phosphatase.

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 4A. ENZYME KINETICS ANALYSIS OF ALKALINE PHOSPHATASE

Alkaline phosphatase catalyzes the cleavage of esters of phosphoric acid.

Alkaline phosphatase (AP) cleaves the colorless substrate 4-nitrophenylphosphate (PNPP)

forming the yellow product p-nitrophenol (PNP).

Like many proteins, several factors may affect enzymatic activity. In this experiment, we will test

the impact of pH, substrate concentration and an inhibitor on the AP activity.

We will be based on determination of Vmax and Km for each condition. Since Vmax and KM

can vary independently of each other, the best enzyme activity will be the one in which Vmax is

fastest and KM is lowest. The ratio of Vmax/KM allows for an easy comparison of the calculated

kinetics parameters.

To measure the activity of AP, one can follow the liberation of phosphate or of the other product

released by hydrolysis. The assay can be simplified by using a substrate whose phosphate-free

product is highly colored. In this experiment, we will utilize 4-nitrophenylphosphate as the

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substrate, which upon hydrolysis releases phosphate to genterate 4-nitrophenolate under alkaline

conditions. 4-nitrophenolate has a high molar absorptivity at 405 nm (405 = 18.8 x 103 M-1cm-1).

This experiment will examine the effects of:

1) substrate concentration

2) pH (5, 7, 10)

3) an inhibitor

on the rate of alkaline phosphatase catalyzed hydrolysis of 4-nitrophenylphosphate.

You are ought to review the enzyme kinetics section of your biochemistry textbook. It will give a

good explanation of Michaelis-Menten kinetics, the plots that you will make with your data, the

method for determining Vmax and Km of alkaline phosphatase from your data, and how to

interpret the effects of the inhibitor on your enzyme.

Materials
1. 1 mM PNPP (p-nitrophenylphosphate) in 0.2 M Tris-HCl (pH 8.0);
2. 0.1 M Tricine at pH 5
3. 0.1 M Tris-HCl at pH 7
4. 0.1 M Tris-HCl at pH 10
5. 50 mM Na2HPO4 at pH 10
6. 0.3 mM PNP
7. 1mg/ml Alkaline phosphatase.

Experimental Procedure: Effect of substrate concentration on the reaction velocity.

1. Get 10 mL of a stock solution of 4 mM p-nitrophenylphosphate substrate in 0.05 M

amidiol buffer pH 9.0. Place it in a 50-mL volumetric flask and dilute it to 50 mL with

0.05 M amidiol buffer pH 9.0.

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2. Make 6 serial 1/2 dilutions of this stock by adding 25 mL of the diluted substrate

prepared in step 1 with 25 mL of 0.05 M amidiol buffer pH 9.0. You should end up with

dilutions containing 0.4, 0.2, 0.1, 0.05, 0.025, and 0.125 mM p-nitrophenylphosphate.

3. Make 1.2 mL of alkaline phosphatase in a 1.5 mL microcentrifuge tube.

4. Make 1.2 mL of 8 mM Na2HPO4 in distilled water from the 150 mM Na2HPO4 stock

solution provided.

5. Measure reaction kinetics using the 6 substrate dilutions ( 0.4, 0.2, 0.1, 0.05, 0.025, and

0.125 mM p-nitrophenylphosphate) that you prepared in step 2.

Enzyme assays:

• Measure the rate of the reaction at substrate dilution as presented in the below table 1.

Also measure the rate for the nonenzymatic reaction. Make these measurements "side-by-

side" at each substrate concentration.

• Add the enzyme (to a final concentration 5ug/ml) last and start measuring quickly.

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 Fill in all the missing data in table 1.

Table 1. Example set up of 1-mL reactions for Alkaline Phosphatase kinetics at varying pH=9
Sample # H2O Buffer Buffer, [PNPP], PNPP, ul Vol of 50 A410nm/min
pH ul uM ug/mL AP

10 0 100
10 0
10 20
10 40
10 60
10 80
10 100
10 200
10 500

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 4B. FACTORS AFFECTING ALKALINE PHOSPHATASE ACTIVITY

Enzyme kinetics are affected by several factors like temperature, pH, inhibitors and
others (can you think about another two factors?).
• Temp and pH: Each enzymatic reaction has an optimum pH and optimum
temperature. Extreme temp or pH disrupts enzyme structure and therefore reaction
rate.

• Enzyme inhibitors:

Enzyme inhibitors fall into two broad classes:

1. Irreversible inhibitors: Inhibitors of this class usually cause an inactivating, covalent

modification of enzyme structure
2. Reversible inhibitors: can be divided into two main categories
a. Competitive inhibitors: Compete with substrate for binding to the active site in the
enzyme.
b. Noncompetitive inhibitors: Inhibitor binding site is distinct from the substrate
binding site.

This experiment will examine the:

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1. Effect of inorganic phosphate (Pi) as an inhibitor on AP enzymatic activity.

2. Effect of pH on the AP activity: For most enzymes, pH can influences the catalytic site

directly by altering the charge of the protein in this region. The pH will also affect tertiary

structure of the enzyme, and may also affect the ability of the enzyme to bind the substrate

3.You are asked also to prepare a calibration curve for PNP (Why cannot you use the calibration

curve prepared last week?).

Experiment procedure:

1) Test AP activity at pH 10 at similarly as tested in table 1 (lab 4A) in the absence and in the

presence of 5mM Pi.

2) Test AP activity at pH 5 similarly as tested in table 1 (lab 4A) in the absence of 5mM Pi

Remember to add the enzyme (to a final concentration 5ug/ml) last and start measuring quickly

3) Calibration curve: Measure the absorbance of 0, 0.03, 0.06, 0.09, 0.12, 0.15, 0.3 μmol PNP at

400nm.

400=18,000 (for PNP)

Write a lab report:

Present the enzyme activity results in umole PNP/mg Prot.*min

Plot v versus [S] (concentration of the substrate), and present the graph.

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Plot 1/v versus 1/[S], and determine Km and Vmax (Lineweaver-Burk plot).

Discuss the Vmax and Km results with and without inhibitor.

Which type of inhibition was it?

Discuss the Vmax and Km results at pH 5 and 10. Which one is expected to be optimal? How can

you test the optimal pH?

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