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Life Sciences Curriculum

LSM 1106
Molecular Cell Biology

AY 2018/2019
Semester II
CONTENTS

Minimal Safety Requirements ........................................................................... 2

Introduction
Some points on the laboratory course .................................................................................... 3
Pipettors ................................................................................................................................. 8
Pipette Controller ................................................................................................................. 10
Spectrophotometry ............................................................................................................... 12

Practical 1: pH and Buffers


The Henderson-Hasselbalch equation and buffers .............................................................. 14
The pH meter ....................................................................................................................... 15
Titration of Histidine monohydrochloride and determination of its buffering region ......... 17
Effect of buffer pKa on buffering capacity ........................................................................... 19
Effect of temperature on the pH of a buffer ......................................................................... 20

Practical 2: Quantitative Protein Estimation


Ultraviolet absorbance of protein......................................................................................... 21
Dye binding method ............................................................................................................. 22

Practical 3: Enzyme Kinetics


To plot the absorption spectra of NAD+ and NADH ........................................................... 25
To study the relationship between absorbance and concentration of NADH ...................... 25
To determine the Km and Vmax of lactate dehydrogenase .................................................... 26
Supplementary Information ................................................................................................. 28

1
MINIMAL SAFETY REQUIREMENTS

 Lab coats must be worn at all times in the lab, but removed before leaving
the laboratory

 Full length long pants must be worn as protection against spills

 Suitable covered footwear must be worn to protect against spills; do not


wear sandals or slippers

 Smoking is strictly forbidden

 Long hair should be tied back

 Do not eat, drink or apply cosmetics in the lab (especially nail polish –
flammable solvents)

 Never use an open flame in the vicinity of flammable solvents

 If you have an abrasion or cut on your hands prior to the class, cover it
before you begin work

 Report any accident to the demonstrators immediately

 Never pipette anything by mouth

 Wash your hands thoroughly with soap and water before leaving the lab

 Familiarize yourself with any additional rules relating to the laboratory in


which you are working. Note the location of eye wash points, safety
shower, fire extinguisher and first aid cabinets

2
INTRODUCTION

SOME POINTS ON THE LABORATORY COURSE

GENERAL OBJECTIVES OF THE LABORATORY COURSE

Biochemistry is essentially an experimental discipline, and it is important to obtain proficiency


for its techniques at an early stage if you wish to pursue the subject further. However, even if
later you wish to proceed elsewhere, the skills you will learn in this course will remain useful
to you in that, for the most part, they have become universally used in the other biological
sciences. You will notice that each practical session has its objectives specified. It will be a
matter for your judgment and comment, in conjunction with discussions with the staff, as to
whether the objectives have been fulfilled.

ATTITUDES TO PRACTICAL WORK

It is important to approach the practical in the right spirit, for an early feel for the way biological
substances, cells and tissues are handled, will stand you in good stead. You should aim at doing
as many of the manipulative procedures as possible with your own hands.

To gain the maximum benefit, it is important to discuss any difficulties with your demonstrator
or lecturer.

Try to plan your work ahead as much as possible; this means reading the text of the practical
and anticipating the various steps you have to perform.

The numerical result of an experiment, in isolation, is of course meaningless; the process is


incomplete without interpretation, which is an aspect you should also develop. Expertise in
this may come slowly, but will be guided where possible.

GENERAL SAFETY IN THE LABORATORY

THE GOLDEN RULE IS TO ASK SOMEBODY IN AUTHORITY ABOUT SAFETY


ASPECTS IF YOU HAVE DOUBTS, AND NOT TO PROCEED UNTIL YOU ARE
SURE OF THE SAFETY OF ANY PROCEDURE

 Do not pipette poisonous and corrosive materials by mouth.


 Wash away an irritant from the skin and/or eyes with copious volumes of cold water from
the sink in the first instance. Inform the demonstrator about the incident and seek advice
from him/her about any necessary treatment.
 Any cuts or burns should be similarly reported.
 There must be no drinking or eating in the laboratory.
 Specific safety precautions will be notified or reiterated in the relevant practical sessions.
All are preventable with common sense precautions and a basic appreciation of laboratory
safety.

3
EQUIPMENT AND GLASSWARES PROVIDED

 Pipettors and tips


 Spectrophotometer and cuvettes
 Basic preparative and volumetric glasswares
 Test tubes and racks
 Any other glasswares and equipment needed for the practical sessions

YOUR OWN SUPPLIES

 Notebook with hard covers (to resist spillages)


 Lab Coat
 Goggles
 Fine-tip marker pen (water-resistant)

SOME GENERAL POINTS OF ETIQUETTE AND GOOD LABORATORY


PRACTICE

 Do not crowd round another worker, both for safety reasons and to avoid distraction.
 Keep any article liable to be damaged off the bench. This includes textbooks and clothing.
 Keep raucous behaviour for outside the laboratory.
 Never pour excess reagents back into the original bottles to avoid possible contamination.
Take small quantities for your individual use.
 Replace stoppers from common reagent bottles immediately.
 Put everything back where you find it.
 Dispose of waste appropriately. The following are some general guidelines on the disposal
of different types of wastes:
1. Corrosive wastes - into the specific liquid waste bin indicated.
2. Organic wastes - into the specific liquid waste bin indicated.
3. Solids should go into bins, provided that they are not classified as biological or
sharps waste.
4. Biological waste will be disposed of into specially marked plastic bags. Whereas
the liquid biological waste is disposed into the specific liquid waste bin indicated.
5. Sharps (broken glass, blades and needles) should go into a specially-labelled sharp
disposal box.

This is also an aspect of laboratory safety (see above).

4
UNITS AND SIGNIFICANT FIGURES

The scientific community insists on uniformity of units in the interests of unambiguousness


and ease of interpretation. In general, this course will require SI units. Because some non-SI
units are inordinately useful in biochemistry their continued use is permissible. These are:

Quantity Name Symbol


minute min
time
hour h
litre l or L*
volume millilitre ml or mL*
microlitre l or L*
temperature degree Celsius C
M mol/L
concentration mM mmol/L
g/100ml (w/v) %

*In print, the upper case “L” is often used to avoid confusion between one (“1”) and lower case “l”.

In addition, concentration is sometimes still expressed as g/100 mL (see table above). “%”
also means quantity of substance dissolved in 100 mL solution (not solvent). The concentration
M for mol/L is still often used, especially in scientific journals. The formal is never used for
the constituents of body fluids but can be used for lists of reagents, for the sake of brevity, as
is done in this manual. Do not make up your own units.

w/v and w/w are self-explanatory terms giving the relative proportions of solids and liquids in
reagents.

In particular, never put a plural for a unit, nor a full stop, e.g. mol/L (not mol./L. or mols/L or
any similar combination).

As important as the correct use of units is the choice of significant figures appropriate to the
accuracy and precision of the method. [Note, accuracy refers to how near the result is to the
“true” result; the true result is only ascertainable by a reference method and is a concept of
minimal importance in much of biochemistry, where comparisons over time and/or between
groups or individuals are more important. Precision refers to the repeatability of a result; that
is’ the closeness of a number of measurements on the same material.] If you report a serum
glucose result as say 5.2 mmol/L you are reporting two significant figures, which are the digits
required to show the precision with which a measurement can be made. To report 5.2 implies
a result nearer 5.2 than 5.1 or 5.3, or more exactly, you believe the result is between 5.15 and
5.25. Were you to report, say, 5.23 mmol/L, this would imply a result between 5.225 and 5.23,
which already seems optimistic for a biological assay. Zeros to the right of the decimal point
are significant but those on the left may or may not be. For example, in 0.003 and 0.0030 the
zeros are meaningful but in 3000, the digit 3 may be the only significant figure, implying a

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result somewhere between 2,500 and 3,500. This difficulty is removed by using exponents, for
example 3.0 x 103 specifies two significant figures.

The most skilled operator dealing with biological substances can rarely achieve repeatability
better than 3% - 5%. Therefore, your choice of significant figures should be appropriate to this.
This may be a difficult concept at first but at a minimum it is important not to be deceived by
the capacity of electronic calculators. Lists of recommended significant figures for substances
in human body fluids are published by authorities in the field and can always be consulted if
need be. An example is the excerpt below.

Examples of Conversions to systéme international (SI) Units

System Component Present Present Unit Conversion SI SI Unit Significant Suggested


† Reference Factor Reference Symbol § Digits Minimum
Intervals Intervals ‡ Increment
(Examples)
P Acetaminophen
Therapeutic 02-0.6 mg/dL 66.16 13.40 mol/L XX 1 mol/L
Toxic 5.0 mg/dL 66.16 300 mol/L XXO 10 mol/L
B, S Acetone Negative mg/dL 172.2 Negative mol/L XXO 10 mol/L
S Alanine 0.35 (37C) Units/L 1.00 0.35 U/L XX 1 U/L
aminotransferase Karmen 0.482  U/L XX 1 U/L
(ALAT) units/mL 0.482  U/L XX 1 U/L
Reitmann-
Frankel
units/mL
S Albumin 4.0-6.0 g/dL 10.0 40.60 g/L XX 1 g/L
S Aldolase 0.6 (37C) Units/L 1.00 0.6 U/L XX 1 U/L
Sibley- 0.7440  U/L XX 1 U/L
Lehninger
units/mL

NOTEBOOK AND PRESENTATION OF RESULTS

It is essential to record as you go along and not rely on memory. There is scope for creativity
in all laboratory work but at this stage it lies mainly in your personal report.

It should however always be headed with the title of the experiment and the date. The practical
instructions should NOT be copied down in the report. Instead you should note your results,
showing clearly how you worked them out, prepare charts where appropriate, and relate any
difficulties or comments.

You are free to make suggestions as to improvements or even to question the rationale,
objectives and conduct of the session.

PRESENTATION OF BIOCHEMICAL DATA

Experimental data are best presented in the form of a table and/or graph.

Tables and graphs must


 have an informative title
 be numbered with Arabic numbers, e.g. Table 1, Figure 2
 have labels for the columns and axes with the quantity and units used in the experiments.
All measurements should be in SI units. The units used should be adjusted so that the
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smallest number of digits is used, e.g. a concentration of 0.0045 mol.L-1 is expressed as 4.5
under the heading “Concentration (mol.L-1)”. If you have a wide range of values, use
multipliers or exponents e.g. 5.0 x 10-2 or E-02. Please refer to “Units and significant
figures” for a more detailed discussion on this subject.

In addition, all graphs should have


 correct choice of coordinates. In most experiments, the change of one parameter affects
another which is measured. The known quantity is called the independent variable and the
measured quantity the dependent variable. Measurement of absorbance is a common
biochemical technique and the absorbance is dependent on the amount of absorbing
substance. Thus the absorbance is the dependent variable.
 In all graphs, the independent variable should be plotted on the horizontal (x) axis (the
abscissa) and the dependent variable on the vertical (y) axis (the ordinate).
 a suitable scale. Select a scale which will accommodate all the data points within the area
of the graph paper. When extrapolation is anticipated, e.g. Lineweaver-Burk plot for
analysis of enzyme kinetics, sufficient space must be left for extension and interception.
 clear, defined symbols to show each determined data point.
 a smooth continuous curve (use a flexible rule) or a straight line by joining all the points
together.

You are strongly encouraged to learn to use software (e.g. Excel) to plot the graphs.

7
PIPETTORS

Figure 1. P1000, P200 and P20 micro-pipettor (from left to right).

In clinical and research laboratories, mouth pipettes have mostly been replaced by specially
designed pipettors. These pipettors are designed to measure and transfer liquids accurately and
safely. They are expensive, which is why you still have to use transfer pipettes for much of
your work. Currently, the Gilson or Eppendorf Pipettors, P10, P20, P200 and P1000 are
provided in the biochemistry laboratory (Figure 1). These numbers represent the maximum
volumes of respectively 10, 20, 200 and 1000 l intended for use on each pipettor. This is
clearly labelled on the push-button.

Pay careful attention to the use of pipettors. You must know how to operate the pipettors
correctly, as it will affect all your subsequent experiments.

OPERATION

1. Ensure that you have chosen the correct volume pipettor, e.g. P10 for 1 to 10 L, P20 for
2 to 20 L, P200 for 20 to 200 L, P1000 for 100 to 1000 L.
2. Setting the volume: The pipettor consists of two or three numbers and is read from top to
bottom. The numbers indicate the volume selected, e.g. 100 is 100 L. The volume is set
by turning the adjustment ring slowly to increase or decrease the volume, making sure not
to overshoot the mark.
3. Press a plastic tip firmly on the shaft of the pipettor to ensure a positive, airtight seal.
4. Aspiration: Press the push-button to the first positive stop. Holding the pipette vertically,
immerse the tip into the sample liquid. Next, release the push-button slowly and smoothly
to aspirate the sample. Wipe the outside of the tip, without touching the orifice.
5. Dispensing: Place the end of the tip against the inside wall of the vessel at an angle. Press
the push-button to the first stop to release the sample and if necessary, press to the second
stop to expel any remaining liquid. Remove the pipettor and release the push-button.
6. Eject the tip by pressing the tip ejector button.
8
PRECAUTIONS AND CARE

 Always press and release the push-button slowly and smoothly.


 Never turn the pipettor upside down.
 Never lay the pipettor on its side when there is liquid in the tip.
 All the above precautions are to prevent liquid from entering the shaft of the pipettor.

9
PIPETTE CONTROLLER
CAUTION: Always use a pipette controller whenever you are working with biological,
caustic or toxic samples. Do not pipette with your mouth.

Figure 2. Pipette Controller.

OPERATION AND PRECAUTION

1. The pipette controller (Figure 2) serves to facilitate the pipetting of liquids with graduated
pipettes, volumetric pipettes and blow-out pipettes of either glass or plastic composition
within the volume range of 0.1 mL to 100 mL. If the instrument is correctly used, the liquid
comes into contact with the pipette only.

2. The pipette controller must not be used with vaporous liquids that are incompatible with
polypropylene (PP), polytetrafluorethylene (PTFE) and silicone.

3. Pipetting:
Compress the suction bellows – Before attaching the pipette,
squeeze the suction bellows.

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4. Attach the pipette:
Hold the pipette as near to its upper end as possible, and
carefully insert it into the adapter until it fits tightly.
Attention: Never use force. Thin pipettes are particularly
liable to break. Avoid the risk of injury!
Once the pipette has been securely attached, always hold the
pipette controller in a vertical position, tip down.

5. Fill the pipette:


Immerse the pipette tip into the liquid. Press the pipetting level
slowly upwards. Fill the pipette so that the level of liquid is
slightly above the required mark.
Attention: Please take care that no liquid enters into the
controller. This would impair the filtering function and reduce
the suction capacity. If liquid does enter the controller, change
the filter. Never turn the pipette upside down or lay the pipettor
on its side when there is liquid within.

6. Adjust the Meniscus:


Use suitable lint-free tissue to wipe the pipette tip. Press the
pipetting lever down slowly, until precise adjustment of the
meniscus has been achieved.

Dispense:
Hold the collecting vessel in an inclined position. Place the
pipette tip against the inner vessel wall. Press the pipetting lever
down to dispense.

In case of pipettes with a waiting time:


(Imprint e.g. “Ex + 15 s”)
As soon as the meniscus in the pipette comes to a standstill, start
waiting time as is indicated on the pipette.
Wipe the pipette tip a few millimetres upward along the wall of
the vessel.

7. In case of blow-out pipettes:


As soon as the meniscus in the pipette tip comes to a standstill,
press the small blow-out bellows to discharge the last drop.
Wipe the pipette tip a few millimetres upward along the wall of
the vessel.
Note: In the case of large pipettes (> 50 mL) the vacuum
contained in the suction bellows is not sufficient to draw in all
the liquid at once. Therefore, squeeze the suction bellows
again and continue drawing up liquid.

8. After pipetting:
Hold the pipette at its extreme upper end, and gently twist and
pull it out of the adapter.
11
SPECTROPHOTOMETRY
QUANTITATIVE LAWS OF LIGHT ABSORPTION

When light passes through a solution of a substance the chance of a quantum of light being
absorbed is proportional to the number of quanta entering the solution, the number of molecules
absorbing substance in the light and to the chance of a quantum being absorbed if it “hits” a
molecule.

This proportionality explains the two empirical laws of light absorption, Beer’s and Lambert’s
Laws. Together they give the Combined Law (Beer-Lambert Law):

log10(I0/I) = A =  × c × l

I0 is the incident light, I is the emergent or transmitted light, A is called the absorbance
(alternatively extinction or optical density),  (the Greek letter epsilon) is the molar absorption
coefficient, c is the molar concentration (mol.L-1), l is the path length in cm.

Note that A is the logarithm of the ratio of two quantities and it has no units.  has the units M-
1
.cm-1 or mol-1.L.cm-1. Note that you also need to specify the wavelength, in nm, which can be
done with a subscript, e.g. A405 is the absorbance at 405 nm.

USE OF THE SPECTROPHOTOMETER

Figure 3. Shimadzu UV-1800 Spectrophotometer.

Spectrophotometers are expensive. Treat them with care.

Currently, the model of spectrophotometers available in the biochemistry laboratory is the


Shimadzu UV-1800 series (Figure 3). Note that the basic operating procedure is the same for
all spectrophotometers. However, each model of instrument has a different set of controls, and
some operations may be performed automatically by the instrument.

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SETTING UP A SPECTROPHOTOMETER

1. Read the instruction card provided on the use of the spectrophotometer.


2. Switch on the instrument.
3. Leave the instrument for a few minutes to stabilize.
4. Key in the wavelength you will be using.
5. Zero the instrument.
(a) Dark or zero transmission setting with no light reaching the detector. This may be done
automatically on some machines.
(b) Place a reference cuvette in the cuvette holder in the light path. This should contain the
solvent with all the buffers and reagents except the substance you wish to measure. This is
a reagent or solvent blank. Autozero the instrument to read zero absorbance (or 100 %
transmission). This step must be performed whenever the wavelength is changed.
5. Now place the cuvette containing the substance you wish to measure in the light path. Read
the absorbance.
6. Remove the cuvettes from the spectrophotometer. Check that you have not spilled any
liquid into or onto the spectrophotometer - if you have, wipe it up immediately. Ask a
demonstrator if it is anything other than plain water.
7. When you have finished leave that lid of the cuvette chamber closed. Switch off the
instrument if no other people would be using it.

Practical points:

Never:
 Stand bottles or cuvettes on top of a spectrophotometer.
 Overfill the cuvettes.
 Mix different types of cuvette in one set of measurements.
 Scratch the optical surfaces.
 Hold the cuvette by the optical surfaces.

Always:
 Check that you have the correct cuvettes:
o Plastic cuvette for visible (320-800 nm) or near UV light. Some plastic
cuvettes (e.g. Bio-Rad trUView cuvettes) are made of material with low UV
absorbance. These cuvettes are suitable for use at both UV and visible
regions.
o Quartz cuvette for far UV (180-320 nm).
for the Shimadzu UV-1800 Spectrophotometer.
 Check that the outside of the cuvette is clean and dry before measuring.
 Wipe it gently with kimwipe.
 Make sure you put the optical, polished faces of the cuvette in the light beam.

13
Practical 1: pH and Buffers

1. OBJECTIVES

1.1 To familiarize yourself with the use of a pH meter.


1.2 To determine the buffering region of a buffer solution.
1.3 To understand the properties of buffers.
1.4 To carry out simple calculations using the Henderson-Hasselbalch equation.

2. INTRODUCTION

The behaviour of most organic compounds is determined by their state of ionization


and hence will depend to a large extent on the hydrogen ion concentration of its
environment. An understanding of the measurement and control of pH is of great
importance in physiology, medicine and biochemical research, since biochemical
reactions are critically dependent on pH. In humans, the pH of both the intracellular and
extracellular compartments are maintained within narrow limits by a combination of
buffers, transport of H+ or HCO3- across the cell membrane, control of blood CO2 and
by excretion of excess acid or base. Similarly, in most biochemical experiments, the
pH of the system is closely regulated by the use of appropriate buffers. The choice of
the buffer is dictated both by its buffering range as well as its compatibility with the
system under investigation.

The Henderson-Hasselbalch equation and buffers

Weak acids such as CH3COOH or KH2PO4 are only partially dissociated in solution.
When titrated with a strong base such as NaOH, the pH of the solution changes as a
function of the amount of base added as shown in the figure below.

14
At any point along the titration, the equilibrium can be defined by the equation

Ka
HA H+ + A-

Where Ka is the equilibrium constant and


[A-][H+]
Ka =
[HA]

The protons are mainly hydrated in solution and exist as H3O+, but for simplicity we
shall refer to them as H+. Since by definition, pH = - log10[H+] and pKa = - log10[Ka],
the above equation may be rearranged and expressed in the form of the Henderson-
Hasselbalch equation

pH = pKa + log [A-]


[HA]

It can be seen from this relationship between pH and pKa that when the concentration
of acid [HA] and conjugate base [A-] are equal, pH = pKa.

In addition, you will note that on either side of the pKa, the acid-base mixture resists
change in pH upon addition of acid or base, i.e. it acts as a buffer. Buffer solutions are
therefore mixtures of weak acids and their conjugate base.

The pH meter

Mettler Toledo pH meter.

The pH meter is routinely used in biochemical laboratories for the accurate


measurement of H+ concentration. The measurement relies on a glass membrane which
is permeable to protons but not to other cations. An acid solution of known H+
15
concentration (A) is trapped within this membrane. When placed in the solution being
measured (B), a potential difference will develop across the glass membrane, the
magnitude of which is given by the Nernst equation

E = 1n RT [H3O+]A
ZF [H3O+]B

(R=gas constant, T=absolute temperature, F=Faraday and Z = valency of ion)

This potential difference is measured against a standard reference electrode which is


usually incorporated into the pH electrode. The potential is directly dependent on the
temperature and the instrument has to be corrected for different temperatures using the
temperature control function. On some instruments, this is done automatically by a
resistance thermometer in the solution (automatic temperature control).

Besides this temperature-dependent variation, the electrode may also vary in efficiency
giving less or even greater potential than that predicted by theory. The scale must
therefore be adjusted to read correctly over the range of pH values being measured.
From this you will realize that the pH meter needs to be calibrated regularly before use.
This can be done using standard buffers of known pH.

Use of the pH meter

Before proceeding to use the pH meter, please consult a demonstrator.

Read carefully the instructions on the use of the pH meter which are available near the
instrument. The glass bulb of the pH electrode is extremely fragile and should be
handled with care at all times. Adhere to the following precautions when using the pH
meter.

1. Ensure that the electrodes are always left with the bulb immersed in distilled
water or a suitable buffer.
2. Both before and after use, rinse the electrodes carefully by dipping into a beaker
of distilled water and then squirting with distilled water from a wash bottle. To
prevent diluting the sample, remove excess water from the electrode by blotting
lightly (but not wiping) with a tissue.
3. When taking measurements, check that the bulb of the electrode is completely
immersed in the solution.
4. Remember to calibrate the pH meter with the standard buffers provided before
use. The steps for calibrating the pH meter are as follows: enter the calibration
mode and place the electrode in standard buffer pH 4. Standardize the pH meter
to read exactly 4. Repeat this process to re-standardize the pH meter with
buffers of pH 7 and pH 10 respectively. Exit the calibration mode before using
the pH meter for your experiments.

16
3. EXPERIMENTS

3.1 Titration of Histidine monohydrochloride and determination of its


buffering region

Histidine is a commonly occurring amino acid present in most proteins and


enzymes. The pKa of its imidazole group ranges from 6-7 depending on the
temperature, ionic strength and the nature of other functional groups present in
its vicinity. At physiological pH, it can readily accept or donate protons. It
plays an important role not only in the buffering action of proteins but also in
the catalytic activity of many enzymes.

Principle: Titration, a graual addition of an acidic solution to a basic solution


or vice versa, can be used to determine the number of acidic or basic groups in
an unknown compound. A specific concentration of the compound is titrated
with a known concentration of acid or base until the equivalence point has been
reached. A graph can then be constructed on which the pH at regular intervals
is plotted along one axis and the number of moles of added acid or base at these
intervals along the other axis. Such a plot is called a titration curve and is usually
sigmoid (S-shaped), with the inflection point - where the curve changes
direction - corresponding to the equivalence point. From the pH at the
equivalence point, the dissociation constant of the acidic or basic group can be
determined. If a compound contains several different acidic or basic groups, the
titration curve will show several sigmoid-shaped curves like steps and the
dissociation constant of each group can be obtained from the pH at its
corresponding equivalence point.

You will perform the titration of histidine solution with NaOH. The titration
curve obtained will be used to determine the pKa and effective buffering range
of histidine.

3.1.1 Materials
 Burette
 Retort stand
 Glass funnel
 pH meter
 Magnetic stirrer

3.1.2 Reagents
 Histidine monohydrochloride (given as dry salt,
MW = 209.63)
 0.05 M NaOH

3.1.3 Procedure
1. Each group will be provided with a 50 mL falcon tube containing
histidine monohydrochloride salt. The mass of the salt is indicated
on the tube. Calculate the volume of deionized water required to
prepare 20 mM histidine monohydrochloride solution in the tube.
Vortex the contents until the salts are fully dissolved.

17
2. Design an experiment for the titration of histidine with 0.05 M
NaOH. You will use 20 mL of 20 mM histidine
monohydrochloride prepared in step 1 for this purpose. Construct a
table to record your data. The table must include a column for the
values of mole NaOH/mole histidine.
3. Titrate at 0.5 mL increments of NaOH. The initial pH of the
histidine monohydrochloride solution should be around 4. Stop the
titration when the pH of the solution reaches between pH 11 to
11.5.

3.1.4 Data handling and questions


(a) Calculate the number of moles of histidine present in 20 mL of
20 mM histidine monohydrochloride solution.
(b) Plot a graph of pH (y-axis) against the number of moles of NaOH
per mol of histidine (x-axis). The advantage of plotting the graph
in this manner is that it is independent of the volume and
concentrations of the solutions used.
(c) From the graph, determine the pKa values. There are two ways
in which this may be done.
(i) By determining the pH when 50% of the acid has been
converted to the conjugate base i.e. after addition of 0.5
and 1.5 mol of NaOH per mol of histidine.
(ii) By determining the center of symmetry i.e. the point of
inflexion of the graph.
Indicate the pKa values obtained from both methods, and show
clearly how the values were obtained.
(d) At what pH does the acid-base mixture show maximal, buffering
capacity? What do you think is the effective buffering range of
this buffer?
(e) If you have reason to believe that the NaOH has not been
accurately prepared, which of the two methods do you think will
give you a more reliable estimate of the pKa? Give reasons for
your answers.
(f) Using the pKa values that you have obtained from the graph,
calculate the pH of the solution after addition of (i) 5 mL and (ii)
12 mL of NaOH. Compare these values with your experimental
data.
(g) The fully protonated form of histidine has the following
structure:

(i) How many ionisable groups are present in histidine


molecules at the initial pH of the experiment?
18
(ii) Which ionisable groups are responsible for the observed
pKa’s?
(h) Draw the structures of the ionic species of histidine that
participate in cellular buffering. The physiological pH is around
7.2 to 7.4.

3.2 Effect of buffer pKa on buffering capacity

Principle: Apart from using a pH meter and constructing a titration curve,


another common method to indicate the endpoint (the point of complete
neutralization) of a reaction is by using visual indicators. In simple acid-base
titrations a pH indicator (e.g. phenolphthalein) may be used, which turns pink
when a certain pH is reached. Due to the logarithmic nature of the pH curve, the
transitions are generally extremely sharp. A single drop of titrant just before the
endpoint can lead to huge changes in pH, which is reflected by an immediate
colour change in the indicator.

In this experiment, we will make use of Universal Indicator - a blend of different


indicators that exhibits smooth color changes over a wide range of pH values –
to investigate how the buffer’s pKa determines its ability to buffer against acid
or base at initial pH.

3.2.1 Materials
 Test tubes
 Disposable Pasteur pipettes
 Universal pH indicator

3.2.2 Reagents
 0.01 M potassium phosphate buffer, pH 7.0 (pKa 6.8)
 0.01 M Tris-HCl, pH 7.0 (pKa 8.1)
 0.05 M HCl
 0.05 M KOH

3.2.3 Procedure
The buffer solutions provided are commonly used in the biochemistry
laboratory.

Design a simple experiment to study the effect of pKa on the buffering


efficiency of the buffers.

3.2.4 Questions
1. What conclusions can you draw from your experiments?
2. Which of these two buffers would you use if you were studying
the properties of a phosphatase which functions optimally at pH
7.2? Explain your choice.

19
3.3 Effect of temperature on the pH of a buffer

Principle: Temperature affects the pH of a buffer by altering the dissociation


constants of the buffer molecules. Depending on the type of buffer, the pH either
increases, decreases, or does not change when the solution is taken from room
temperature to 4ºC (i.e. the temperature of a cold room, refrigerator, or ice bath).
This temperature dependent behavior is most critical for protein or enzyme
purification schemes in which both the pH and temperature of the procedure are
critical to the isolation of an active form of the protein.

3.3.1 Materials
 Ice box
 pH meter

3.3.2 Reagents
 0.01 M potassium phosphate buffer, pH 7.0
 0.01 M Tris-HCl, pH 7.0

3.3.3 Procedure
Measure the pH of the 0.01 M Tris-HCl and 0.01 M potassium
phosphate buffer provided at room temperature and after cooling to 4C
by placing the solutions in an ice bath.

3.3.4 Questions
What effect does temperature have on the pH of the Tris-buffer and
potassium phosphate buffer? Give an explanation for your observation.

20
Practical 2: Quantitative Protein Estimation

1 OBJECTIVES

1.1 To record a protein spectrum and to estimate the protein concentration using a
direct spectrophotometric method.
1.2 To estimate the protein concentration using a dye-binding (“Bradford”) method.

2 INTRODUCTION

Proteins are ubiquitous components of all living tissues, whether of animal, plant or
bacterial origin. They serve indispensable functions in cellular architecture, catalysis,
regulation, contractile processes and immune defence. Proteins are intimately
concerned with virtually all physiological events.
Protein estimation is therefore very important in every investigation in biochemistry.
For example, laboratory practice in protein purification often requires a rapid and
sensitive method for the quantitation of protein. Presently, a variety of methods are
available for the determination of protein concentration of a given sample. The methods
employ different principles, and may be sensitive to interferences by certain salts,
buffer components and solvents. Each method therefore has certain unique and useful
characteristics as well as limitations. In the biochemical laboratory, the most widely
used methods often employ photometric and/or colorimetric analyses as these methods
are simple, rapid and have the required sensitivities.

3 EXPERIMENTS

3.1 Ultraviolet absorbance of protein

Principle: A common method of protein estimation depends on the


measurement of the optical density of a protein solution. Proteins exhibit UV
absorption spectra with a maximum at 280 nm, and this is due primarily to the
tyrosine and tryptophan content of the protein. A pure protein can therefore be
quantitated by measuring its absorbance at 280 nm in a spectrophotometer. The
method is often used for a rough measurement of protein concentration because
it is very quick and simple. Its outstanding advantage is that it is non-destructive
and therefore the protein solution can be recovered completely.

3.1.1 Materials
 5 mg/mL bovine serum albumin (BSA)
 Two samples of unknown concentration

3.1.2 Procedure
1. Prepare 10 mL of 1.0 mg/mL BSA from the 5 mg/mL BSA stock,
using water as the diluent.
2. Construct absorption spectra for the 1.0 mg/mL BSA and the two
unknowns by measuring their absorbances at 10 nm intervals over

21
the range 250-360 nm (start measurement from the longer
wavelength first).
3. Calculate the concentrations of all 3 samples using the respective
absorbance values at 280 nm.

A rough conversion is

Protein in mg/mL = E280 (absorption at 280 nm in a 1 cm cuvette)

This is strictly true only for proteins with E1% (absorption of a 1%


protein solution) values of 10. As the absorption at 280 nm depends
on the tyrosine and tryptophan content of the protein, each protein
will have a different E1%. Tables giving the E1% of many proteins
can be found in the literature. [For BSA, the value is 6.67].

3.1.3 Questions
1. Calculate the protein concentration of the prepared BSA and
unknown samples using E1% values of 10.0 and 6.67.
2. What can you surmise by comparing between the absorption spectra
of the 1 mg/mL BSA and the two unknowns?

3.2 Dye binding method

Principle: These methods are based on the noncovalent binding of dyes to


proteins causing a colour change which may be measured in a
spectrophotometer. The most widely used of these methods is that of Bradford
using Coomassie Brilliant Blue G-250, now marketed commercially (Bio-Rad)
as a protein assay kit. In this method, there is a shift in the absorption maximum
of an acidic solution of Coomassie Brilliant Blue G-250 from 465 nm to 595
nm on binding to protein, and the assay measures the increase in absorption at
595 nm.

3.2.1 Materials
 5 mg/mL bovine serum albumin (BSA)
 Samples of unknown concentration
 Dye reagent concentrate

3.2.2 Procedure
1. The linear range of the assay for BSA is 0.1 to 0.8 mg/mL.
2. Prepare five (5) dilutions of BSA within this range from the 1.0
mg/mL BSA prepared earlier, using water as diluent.
3. At the same time, also dilute the unknown samples to within this
linear range by referring to their concentrations estimated in part 1.
4. Pipette 50 L of each standard and unknown sample solution into a
clean, dry tube. Protein solutions are normally assayed in duplicate
or triplicate.
5. For the blank or control, pipette 50 L of water into the tube instead.

22
6. Dilute 1 part Dye Reagent with 4 parts distilled water. Add 2.5 mL
of diluted dye reagent to each tube and vortex. Incubate at room
temperature for at least 5 minutes and not more than one hour.
7. Measure the absorbance at 595 nm, and plot a standard calibration
curve of absorbance against protein (BSA).
8. Estimate the protein concentration of the unknown samples from
their absorbances at 595 nm using the calibration curve.

3.2.3 Questions
1. Construct a protein standard curve based on the dye-binding
(“Bradford”) method, and establish the protein concentration of the
unknown solutions provided.
2. Compare the protein concentration of the unknowns obtained from
the two methods. Comment on the advantages and disadvantages of
the two methods in estimating the protein concentration of an
unknown sample. Which method will provide a more reliable
estimate of the protein concentration in our experiment? Use the
experimental data obtained to support your argument.
4. Calculate the molar extinction coefficient of BSA if a 1.0 mg/mL
solution in buffer has an absorbance of 0.667, and the molecular
weight of BSA is 66,000.

23
Practical 3: Enzyme Kinetics

1. OBJECTIVES

1.1 To plot the absorption spectra of NAD+ and NADH.


1.2 To understand the rationale for measuring reactions of NAD+-linked
dehydrogenases at 340nm.
1.3 To examine the relationship between A340 and concentration of NADH and to
determine the molar absorption coefficient of NADH.
1.4 To understand how initial velocity of an enzyme-catalysed reaction is obtained.
1.5 To plot the Michaelis-Menten curve and Lineweaver-Burk plot with the data
obtained.
1.6 To calculate the values of Km and Vmax of LDH.

2. INTRODUCTION

To obtain Michaelis-Menten kinetic data for an enzyme, it is necessary to control all


variables except the substrate concentration. When the initial velocity is plotted against
the substrate concentration, one will have the Michaelis-Menten curve, from which,
however, a determination of Km and Vmax is difficult. Therefore, Lineweaver-Burk plot
is often used to obtain these Michaelis-Menten kinetic data. In addition, this plot can
also be used to characterize and measure the effects of inhibitors of an enzyme. Lactate
dehydrogenase (LDH) will be used in this practical to illustrate how such kinetic data
are obtained. LDH catalyses the following reaction:

Lactate + NAD+  pyruvate + NADH + H+

Many NAD+- and NADP-linked dehydrogenases can be measured


spectrophotometrically by monitoring the absorbance of NADH or NADPH formed.
The activities of other enzymes may also be determined in a similar way if the products
of the catalysed reactions can be coupled to those catalysed by dehydrogenases. In this
practical, the production of NADH will be monitored to illustrate the application of
spectrophotometry for studying enzyme kinetics.

3. EXPERIMENTS

3.1 To plot the absorption spectra of NAD+ and NADH

3.1.1 Materials
 0.1 mM solution of NAD+, pH 7.5
 0.1 mM NADH, pH 9.0.

3.1.2 Procedure
Record the absorbance of each solution between 240 to 400 nm at
intervals of 20 nm. With every change in wavelength setting, zero the

24
instrument with a blank containing buffer alone. Why is it necessary to
zero the instrument?

3.1.3 Report and Questions


1. Plot a graph to show the relationship between absorbance (y-
axis) against wavelength (x-axis).
2. What is the main difference in the absorption spectra between
NAD+ and NADH?
3. What is the wavelength of choice for measuring NADH in a
mixture containing both NAD+ and NADH? Give your reasons
for your answer.

3.2 To study the relationship between absorbance and concentration of NADH

3.2.1 Materials
 1 mM NADH in 0.05 M sodium pyrophosphate buffer, pH 9.0
 0.05 M sodium pyrophosphate buffer, pH 9.0

3.2.2 Procedure
Prepare a series of tubes containing final concentrations of 0.01 mM to
0.2 mM NADH from 1 mM NADH solution, using the 0.05M sodium
pyrophosphate buffer, pH 9.0 as the diluent. The final volume of each
diluted solution should be 3 mL. Read the absorbance of NADH for each
concentration at 340 nm.

3.2.3 Report
1 Tabulate your data in the form of a table.
2 Plot a graph to show the relationship between absorbance and
concentration of NADH.
3 Calculate the molar (or millimolar) extinction coefficient of NADH,
applying the Beer-Lambert equation.

3.3 To determine the Km and Vmax of lactate dehydrogenase

3.3.1 Materials
 200 mM sodium lactate
 10 mM NAD+
 2 units/mL LDH
 0.05 M sodium pyrophosphate buffer, pH 9.0

3.3.2 Procedure
Pipette 0.1 mL NAD+ and 30 l of sodium lactate into a cuvette and
topped it up with buffer to a final volume of 2.8 mL. Zero the
spectrophotometer. Start the reaction by adding 0.2 mL of LDH. Cover
the cuvette with a small piece of parafilm and mix rapidly and
thoroughly by inversion. Monitor the progress of the reaction by
recording A340 at 0.5 min intervals for 4 min. Repeat the above assay
with different volumes (up to 300 L) of sodium lactate, keeping other
25
conditions unchanged. You should have 5 different concentrations of
sodium lactate (final lactate concentration ranges from 2 to 20 mM).

3.3.3 Report
1 Plot the increasing absorbance against the reaction time for each
substrate concentration. Find the initial velocity from the curves
obtained (note: that the value obtained here will be in absorbance
units per min)
2 By using the information from 3.2.3, convert the initial velocity to
moles per min.
3 Plot the Michaelis-Menten curve and Lineweaver-Burk plot (note:
in both plots, the initial velocity should be in moles per min).
Compare and comment on the Km and Vmax values obtained from
these two procedures.
4 What are the advantages and disadvantages of the Michaelis-
Menten curve and the Lineweaver-Burk plot?

26
Practical 3: Enzyme Kinetics

3.1 To plot the absorption spectra of NAD+ and NADH

Wavelength (nm) NAD+ NADH


400
380
360
340
320
300
280
260
240

3.2 To study the relationship between absorbance and concentration of NADH

mM NADH 1 mM NADH 0.05 M buffer pH Total Volume A340


(mL) 9.0 (mL) (mL)
0.00 3
0.01 3
0.05 3
0.10 3
0.15 3
0.20 3

3.3 To determine the Km and Vmax of lactate dehydrogenase

Tube mM lactate 200 mM 0.05 M 10 mM LDH Total reaction


No. (final conc) lactate (mL) buffer pH 9.0 NAD+ (mL) (mL) volume (mL)
(mL)
1 2 0.1 0.2 3
2 4 0.1 0.2 3
3 6 0.1 0.2 3
4 12 0.1 0.2 3
5 20 0.1 0.2 3

Time A340
(min) Tube 1 Tube 2 Tube 3 Tube 4 Tube 5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0

27
Supplementary Information
for
Practical 3: Enzyme Kinetics
Coenzymes:
NAD+: Nicotinamide adenine dinucleotide, oxidized form
NADH: Nicotinamide adenine dinucleotide, reduced form
NADPH: Nicotinamide adenine dinucleotide phosphate, reduced form

(NADP+: Nicotinamide adenine dinucleotide phosphate, oxidized form)

28
An illustration of how NADPH is utilised in metabolism.

The free energy released during metabolism is conserved by the synthesis of ATP from ADP
and phosphate or by the reduction of the coenzyme NADP+ to NADPH. ATP and NADPH
are the major free energy sources for anabolic pathways.

29