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Arrhenius's concept: An acid is a substance which An acid dissociation constant, Ka, (also known as acidity
dissociates in aqueous solution, releasing the hydrogen constant, or acid-ionization constant) is a quantitative measure
ion H+ (a proton): of the strength of an acid in solution. It is the equilibrium
HA A− + H+ constant for a chemical reaction known as dissociation in the
The equilibrium constant for this dissociation reaction is context of acid-base reactions. The equilibrium can be written
known as a dissociation constant. The liberated proton symbolically as:
combines with a water molecule to give a hydronium (or HA A− + H+
oxonium) ion H3O+, and so Arrhenius later proposed that
the dissociation should be written as an acid–base where HA is a generic acid which dissociates by splitting into
reaction: A−, known as the conjugate base of the acid, and the hydrogen
HA + H2O A− + H3O+ ion or proton, H+, which, in the case of aqueous solutions,
Brønsted and Lowry generalized this further to a proton exists as a solvated hydronium ion.
exchange reaction. The dissociation constant is usually written as a quotient of the
acid + base conjugate base + conjugate acid. equilibrium concentrations, denoted by [HA], [A−] and [H+]:
The acid loses a proton, leaving a conjugate base; the
proton is transferred to the base, creating a conjugate [H+] [A−]
acid. For aqueous solutions of an acid HA, the base is Ka= [HA]
water; the conjugate base is A− and the conjugate acid is
the hydronium ion. The Brønsted–Lowry definition
applies to other solvents, such as dimethyl sulfoxide: the Due to the many orders of magnitude spanned by Ka values, a
solvent S acts as a base, accepting a proton and forming logarithmic measure of the acid dissociation constant is more
the conjugate acid SH+. commonly used in practice. pKa, which is equal to −log10 Ka,
The designation of an acid or base as "conjugate" may also be referred to as an acid dissociation constant:
depends on the context. The conjugate acid BH+ of a base
B dissociates according to pKa= -log10 Ka
The larger the value of pKa, the smaller the extent of
BH+ + OH − B + H2O dissociation. A weak acid has a pKa value in the approximate
which is the reverse of the equilibrium range −2 to 12 in water. Acids with a pKa value of less than
about −2 are said to be strong acids; a strong acid is almost
H2O (acid) + B (base) OH− (conjugate base) + BH+ completely dissociated in aqueous solution, to the extent that
(conjugate acid). the concentration of the undissociated acid becomes
undetectable. pKa values for strong acids can, however, be
The hydroxide ion OH−, a well known base, is here estimated by theoretical means or by extrapolating from
acting as the conjugate base of the acid water. Acids and measurements in non-aqueous solvents in which the
bases are thus regarded simply as donors and acceptors of dissociation constant is smaller, such as acetonitrile and
protons respectively. dimethylsulfoxide.
Water is amphiprotic: it can react as an acid or a base. In particular, the pH of a solution can be predicted when the
Another example of an amphiprotic molecule is the analytical concentration and pKa values of all acids and bases
bicarbonate ion HCO3− which is the conjugate base of the are known; conversely, it is possible to calculate the
carbonic acid molecule H2CO3 in the equilibrium equilibrium concentration of the acids and bases in solution
when the pH is known.
H2CO3 + H2O HCO3− + H3O+ pH: pH is a measure of the acidity or basicity of a solution. It
but also the conjugate acid of the carbonate ion CO32− in is defined as the cologarithm of the activity of dissolved
(the reverse of) the equilibrium hydrogen ions (H+). Hydrogen ion activity coefficients cannot
be measured experimentally, so they are based on theoretical
HCO3− + OH− CO32− + H2O. calculations. The pH scale is not an absolute scale; it is
Carbonic acid equilibria are important for acid-base relative to a set of standard solutions whose pH is established
homeostasis in the human body. by international agreement.
Henderson–Hasselbalch equation
pH is defined as minus the decimal logarithm of the
hydrogen ion activity in an aqueous solution. By virtue of
its logarithmic nature, pH is a dimensionless quantity. pH = pKa – log [AH]/[A-]
pH= - log aH = log10 1/aH
where aH is the (dimensionless) activity of hydrogen ions.
The reason for this definition is that aH is a property of a
single ion which can only be measured experimentally by
means of an ion-selective electrode which responds,
according to the Nernst equation, to hydrogen ion activity.
pH is commonly measured by means of a combined glass
electrode, which measures the potential difference, or
electromotive force, E, between an electrode sensitive to
the hydrogen ion activity and a reference electrode, such
as a calomel electrode or a silver chloride electrode. The
combined glass electrode ideally follows the Nernst
equation:
Eo - E
E = Eo + RT/nF loge (aH); pH = 2.303 RT/F
At half-neutralization [AH]/[A−] = 1; since log(1) =0 , the pH
at half-neutralization is numerically equal to pKa. Conversely,
where E is a measured potential , Eo is the standard
when pH = pKa, the concentration of AH is equal to the
electrode potential, that is, the electrode potential for the
concentration of A−.
standard state in which the activity is one. R is the gas
constant T is the temperature in Kelvin, F is the Faraday The buffer region extends over the approximate range pKa ± 2,
constant and n is the number of electrons transferred, one though buffering is weak outside the range pKa ± 1. At pKa ±
in this instance. The electrode potential, E, is proportional 1, [AH]/[A−] = 10 or 1/10.
to the logarithm of the hydrogen ion activity.
If the pH is known, the ratio [AH]:[A−] may be calculated.
This definition, by itself, is wholly impractical because the
This ratio is independent of the analytical concentration of the
hydrogen ion activity is the product of the concentration
acid.
and an activity coefficient. The single-ion activity
coefficient of the hydrogen ion is a quantity which cannot Buffer solution of a desired pH can be prepared as a mixture of
be measured experimentally. To get round this difficulty a weak acid and its conjugate base. In practice the mixture can
the electrode is calibrated in terms of solutions of known be created by dissolving the acid in water, and adding the
activity. requisite amount of strong acid or base. The pKa of the acid
must be less than two units different from the target pH.
pH in living systems
Polyprotic acids are acids that can lose more than one proton.
Compartment pH The constant for dissociation of the first proton may be denoted
as Ka1 and the constants for dissociation of successive protons
Gastric acid 0.7 as Ka2, etc. Phosphoric acid, H3PO4, is an example of a
Lysosomes 4.5 polyprotic acid as it can lose three protons.
TYPES OF ERRORS
Experimental errors can be broadly classified into
two categories:
a. Systemic errors and
b. Random errors
When an error affects all measurements in the same
way it is called a systemic error. In most cases, the
cause of this error is known and introducing a
correction factor can minimize the error. For
example, a watch showing an error of + five minutes
(five minutes fast). In this case we can reduce five
minutes from the time shown by the clock to get the
correct time. A balance that shows an error of – 0.5
gm can be adjusted for that error effectively if the
fact is known. If an error occurs due to unknown
reasons it is called a random error or an accidental
error. This type of error can be detected by repeating
the experiments under the same conditions. If
different experimental values or results when
repeating the experiments without changing the
experimental conditions are found,
then there are random errors. These errors can be
quantified and minimized by applying methods of
statistical analysis.
The results or data of an experiment should be
reliable and reproducible. The term precision refers
to the reliability and reproducibility of results. It
also indicates the magnitude by which the data is
free from random errors. We also use the term
accuracy to refer to the quality of the data. When
there is a minimum of both systemic and random
errors or when it is almost zero and the results are
reproducible, then we refer to the data as accurate.
Liquid chromatography of biomolecules
Proteins, peptides, DNA, RNA, lipids, and organic
cofactors have various characteristics such as electric
charge, molecular weight, hydrophobicity, and surface
relief. Purification is usually achieved by using methods
that separate the biomolecules according to their
differences in these physical characteristics, such as ion
exchange, gel filtration, and affinity chromatography.
Ion-Exchange Chromatography of Proteins
In ion exchange chromatography, the stationary solid
phase commonly consists of a resin with covalently
attached anions or cations. Solute ions of the opposite
charge in the liquid, mobile phase are attracted to the
ions by electrostatic forces. Adsorbed sample
components are then eluted by application of a salt
gradient which will gradually desorb the sample
molecules in order of increasing electrostatic
interaction with the ions of the column (Figs. 1– 2).
Because of its excellent resolving power, ion
exchange chromatography is probably the most
important type of chromatographic methods in many
protein preparations.
The choice of ion exchange resin for the purification
of a protein largely depends on the isoelectric point,
pI, of the protein. At a pH value above the pI of a
Fig. 1 Example of ion exchange chromatography. (a)–
protein, it will have a negative net charge and adsorb
(c) Loading the column: mobile anions (or cations) are
to an anion exchanger. Below the pI, the protein will
held near cations (or anions) that are covalently attached
adsorb to a cation exchanger. For example, if the pI is
to the resin (stationary phase). (d)–(f) Elution of the
4 then in most cases it is advisable to choose a resin
column with a salt gradient: the salt ions weaken the
which binds to the protein at a pH > 4. Since at pH > 4
electrostatic interactions between sample ions and ions of
this protein is negatively charged, the resin has to be
the resin; sample molecules with different electrostatic
an anion ion exchanger, e.g., DEAE. One could also
properties are eluted at different salt concentrations,
use a pH < 4 and a cation exchanger, but many
typically between 0–2 M. (g) Interaction of sample
proteins are not stable or aggregate under these
molecules with ions attached to the resin: at a suitable
conditions. If, in contrast, the protein we want to
pH and low salt concentration, most of the three types of
purify has a pI = 10, it is positively charged at usually
biomolecules to be separated in this example reversibly
suitable conditions for protein ion exchange
bind to the ions of the stationary phase.
chromatography, i.e., at a pH around 7. Thus, in
general for this protein type we have to choose a
cation ion exchange resin, e.g., CM, which is
negatively charged at neutral pH.
The capacity of the resin strongly depends on the pH
and the pI of the proteins to be separated (Fig. 4;
Table), but also on the quality of the resin, the applied
pressure, and the number of runs of the column
(Fig.5). To improve the life of the resin, it should be Fig. 2 Two ion exchangers: diethyl-amino-ethyl (DEAE)
stored in a clean condition in the appropriate solvent and carboxy methyl (CM). The positive charge of DEAE
and not be used outside the specified pH range and attracts negatively charged biomolecules. CM is suitable
pressure limit. for purification of positively charged biomolecules
Fig. 3 Example for the salt concentration during
adsorption of a sample to an ion exchange column,
subsequent elution of the sample, and cleaning of the
column. Example of a purification protocol: First the
solution of biomolecules and impurities in buffer
contained in a syringe is loaded onto the column. The
biomolecules and some of the impurities bind to the
ions attached to the resin. Loading is completed and
non-binding molecules are partly rinsed through the
column with some further buffer. The next step is to
apply a salt gradient with a programmable pump which
mixes buffer with extra salt containing buffer. The
steep salt gradient at the beginning elutes most of the
weakly binding impurities. At a certain salt
concentration, the biomolecules to be purified elute
from the column. Elution is monitored with an
absorption detector at 280 nm wavelength and the
sample fraction collected. After each run the column is
cleaned with 1–2 M KCl. This removes most of the
strongly binding sample impurities
The stationary phase consists of semi-permeable, porous Proteins are separated roughly according to their molecular
beads with a well-defined range of pore sizes. The semi- weight because this is the major contribution to molecular size.
permeable porous beads are crosslinked polymers. However, the shape will affect its apparent size in solution.
Degree of crosslinking is controlled carefully to yield different Hence, gel filtration is NOT recommended for separating proteins
pore sizes. The stationary phase is said to have a with only a small difference in molecular weight.
fractionation range (due to the different pore sizes), meaning
Determination of molecular weight
that molecules within that molecular weight range can be
separated. Porous material must swell up and imbibe/absorb Consider the separation of a mixture of i. glutamate dehydrogenase
the liquid phase. The created solvent-filled ‘sponge’ allows (MW 290,000),lactate dehydrogenase (MW 140,000), serum albumin
diffusion of molecules. Therefore, stationary phase may be (MW 67,000), ovalbumin (MW 43,000), and cytochrome c (MW
hydrophilic to imbibe aqueous media, or lipophilic to imbibe 12,400) on a gel filtration column: fractionation range 15,000 -
non-polar organic solvents. 150,000).
When the protein mixture is applied to the column, glutamate
Mobile Phase
dehydrogenase would elute first because it is above the upper
The mobile phase contains a mixture of solutes. Small solutes fractionation limit. Therefore it is totally excluded from the inside of
will diffuse in and out of the pores (obeying Fick’s law). Their the porous stationary phase and would elute with the void volume
path through the column is longer and The elution time will be (V0). Proteins larger than the exclusion range of the resin are unable
longer to enter the pores and pass quickly through the column in the spaces
Chromatogram between the resin. This is known as the void volume of the column.
Cytochrome c is below the lower fractionation limit and would be
completely included, eluting last. The other proteins would be
partially included and elute in order of decreasing molecular weight.
Reproducible Polymerization:
Reproducible polymerization is one of the most
important ways to ensure that your samples migrate
as sharp, thin bands to the same location in the gel
every time. Attention to polymerization will also
help keep the background of your stained gels low. Figure : Log of the molecular weight (in daltons) of a
Acrylamide polymerization is affected by the protein versus the relative mobility. Reproduced with
amount of oxygen gas dissolved in the solution, the permission from Bio-Rad Laboratories.
concentrations and condition of the catalysts, the
temperatures and pH of the stock solutions, and the Straight % Gel or a Gradient Gel?
purity of the gel components. If you want to resolve proteins that are within a few
thousand daltons of each other in molecular weight,
Gel Percentage vs. Catalyst Concentration
then use a straight percent gel (the same concentration
of acrylamide throughout the gel). To get baseline
resolution for such proteins, that is, to get clear,
unstained space between bands, you may need to use a
longer gel. Mini gels have 6 to 8 cm resolution space.
WHICH GEL SHOULD YOU USE? SDS-PAGE, Large gels have 12 to 20cm space. The closer the
NATIVE PAGE OR I SOELECTRIC FOCUSING? bands are in molecular weight, the longer the gel must
The strategy you choose depends on your goal, of be.
course. If you want to determine the molecular weight A gradient gel is used to resolve a larger molecular
of your protein, use SDS-PAGE. If you want to weight range than a straight percent gel. A 10% gel
measure the isoelectric point of your protein, choose resolves proteins from 15 to 100kDa, while a 4% to
isoelectric focusing (IEF). For proteomics work, use 2- 20% gradient gel resolves proteins from 6 to 300kDa,
D electrophoresis (IEF followed by SDS-PAGE). although the restriction about good molecular weight
Native PAGE is used to assay enzyme activity, or other determination discussed above still holds. Accurate
biological activity, for example, during a purification molecular weights can be determined with gradient
procedure. Each kind of protein PAGE has issues to gels
consider, and these issues are addressed in the next
section.
Isoelectric Focusing: Location of Band of Interest
Isoelectric focusing (IEF) measures the Sample proteins move in a native gel as a function of
isoelectric point, or pI, of a protein. The main their charge as well as their mass and conformation,
problem for IEF is sample solubility, seen as and because of this, the location of the protein band of
interest may be difficult to determine. For instance, in
streaking or in-lane background on the stained
some buffer systems, BSA,at 64kDa, will move in
IEF gel, or horizontal streaking on a 2-D gel. front of soybean trypsin inhibitor, at 17kDa.The easiest
Sample solubilization should be way to detect the protein of interest is to determine
optimized for each new sample. At present its location by Western blotting. Alternatively, the
there are two kinds of IEF gels in use: gels protein’s location can be monitored by enzyme activity
formed with carrier ampholytes, and gels or bioassay, which usually requires elution from the
formed with acrylamido buffers, known as gel.
IPG gels (immobilized pH gradient gels).
How Can You Be Sure That Your Proteins Have
Sufficient Negative Charge to Migrate Well into a
Native PAGE Gel?
To determine this, it is useful to have some idea of the
pI of the protein of interest. The pH of the buffer
should be at least 2 pH units more basic than the pI of
the protein of interest. An alternative is to use an acidic
buffer system, and reverse the polarity of the
electrodes. This works well for very basic proteins.
Figure: 2-dimensional electrophoresis. A protein mixture is first separated on an IEF-gel, which is then
mounted across a Laemmli-gel. The second electrophoresis separates the bands of proteins with identical pI
by molecular weight. About 1000 different proteins can be distinguished on a 2d-gel of mammalian cells.
Theory of microscopy
10-14 10-12 10-10 10-8 10-6 10-4 10-2 1 102 104 106 108
Wavelength in Meters
1010 108 106 104 102 1 10-2 10-4 10-6 10-8 10 -10 10-12 10-14
1. Natural oxygen contains three isotopes with atomic masses in amu of 15.9949, 16.9991, and 17.9992
and relative abundances of 2500:1:5. Determine to three decimal places the average atomic mass of
oxygen.
2. Determine the energy required to move an electron from the K to the L shell in tungsten and in
hydrogen, and explain the difference.
3. How many MBq of132I (T1/2=2.3 hours) should be ordered so that the sample activity will be 500 MBq
when it arrives 24 hours later?
4. . How many atoms and grams of 90Y are in secular equilibrium with 50 mCi of 90Sr?
5. If a radionuclide decays for an interval of time equal to its average life, what percentage of the original
activity remains?
Measurement of Dose The sensitivity of the system depends on the voltage
When alpha or beta particles, or gamma radiation, pass applied between the electric plates. Since alpha
through matter, they form ions. They accomplish this particles are significantly easier to detect than beta
by knocking electrons from the orbits of the molecules particles, it requires lower voltage to detect the high
they pass through. We can monitor the ionization effect energy alpha particles. In addition, alpha particles will
by allowing the radiation to pass through dry air and penetrate through the metal casing of the counter tube,
measuring the numbers of ions formed. This is most whereas beta particles can only pass through a quartz
often done by designing a chamber with an electrical window on the tube. Consequently, ionization chambers
charge capacitance, allowing the radiation to pass are most useful for measuring alpha emissions. High-
through the chamber and monitoring the amount of energy beta emissions can be measured if the tube is
capacitance discharge caused by the formation of ions. equipped with a thin quartz window and the distance
The device is a Geiger-Mueller Counter and has many between the source of emission and the tube is minimal.
variations. The ionizing ability is measured in A modification of the basic ionization chamber is the
roentgens, and a roentgen is the number of ionizations pocket dosimeter. This device is a capacitor, which is
necessary to form one electrostatic unit (esu) in 1 cc of charged by a base unit and which can then be carried as
dry air. Since the roentgen is a large unit, dosages for a portable unit. They are often the size and shape of a
cell research use are normally divided into milli- pen and can thus be carried in the pocket of a lab coat.
roentgens (mR). When exposed to an ionizing radiation source, the
Curies measure the amount of radioactive decay, and capacitor discharges slightly. Over a period of time, the
roentgens measure the amount of radiation transmitted charge remaining on the dosimeter can be monitored
through matter, over distance. Neither unit is useful in and used as a measure of radiation exposure. The
determining biological effect, since biological effect dosimeters are usually inserted into a reading device
implies that the radiation is absorbed by the tissues that that is calibrated to convert the average exposure of the
are irradiated. The rad (radiation absorbed dose) is a dosimeter directly into roentgens or rems. Since the
unit of absorbed dose and equals 100 ergs absorbed in 1 instrument works by discharging the built-up charge,
gram of matter. and the charge is on a thin wire in the center of the
dosimeter, it can be completely discharged by the
Detection of Radioactivity flexing of that wire, as it touches the outer shell upon
Ionization chambers. The most common method of impact. When later read for exposure, the investigator
measuring radiation exposure is the use of an will be informed that they have been exposed to
ionization chamber. Among the more common forms of dangerously high levels of radiation, since there will be
ionization chambers are the Geiger-Müller counter, no charge left in the dosimeter. Besides causing great
scintillation counter, and pocket dosimeter. consternation with the radiation safety officer, and a
The chambers are systems composed of 2 electrical good deal of paper work, it also causes some unrest
plates, with a potential established between them by a with the investigator. The dosimeters should be used in
battery or other electrical source. In effect, they a location where they cannot impact any other objects.
function as capacitors. The plates are separated by an Since the dosimeters normally lack the fragile and
inert gas, which will prevent any current flow between vulnerable quartz windows of a Geiger tube, and carry
the plates. When an ionizing radiation enters the lower voltage potentials, they are used for the
chamber, it induces the formation of an ion, which in measurement of x-ray and high energy gamma
turn is drawn to one of the electrical plates. The radiation, and will not detect beta emissions.
negative ions are drawn to the anode (+ plate), while
the positive ions are drawn to the cathode (– plate). As beta
the ions reach the plates, they induce an electric current
to flow through the system attached to the plates. This insulator coated with Ag or graphite “cathode”
is then expressed as a calibrated output, either through
the use of a digital or analog meter, or as a series of Ar+ Ar+ Ar+
Ar0 Ar+ + e- e- e- e-
clicks, by conversion of the current through a speaker.
Tungsten (W) wire “anode”
Expose to film
or emulsion
1. Concentration Determination
This is a direct application of the Beer–Lambert law.
If the absorption coefficient for a molecule is known,
then the concentration of the molecule in solution can
be obtained by taking an absorption spectrum of the
solution and solving the equation:
Fluorescence
Absorption of a photon of appropriate energy causes an
electronic transition from ground state to an excited
state. Once a molecule is in an electronically excited
Fig. Difference spectrum for hydrazone formation state, the energy must eventually be dissipated for the
reaction as a function of time. molecule to return to its ground state.
5. Fluorescence Anisotropy
Fluorophores absorb photons that have their electric
The lifetime of a single electronic transition is
dipoles aligned parallel to the transition moment of
characterized by a single exponential decay of
the fluorophore. When freely diffusing fluorophores
fluorescence and is the time required for the fraction
are excited with polarized light, the emitted light will
of the population of molecules in the excited state to
normally be depolarized as a result of rotational
decrease by a factor of 1/e, or ~ 37%:
diffusion during the lifetime of the excited state. The
extent of depolarization is assessed by determining
the intensity of light emitted parallel (Ik) and
perpendicular (I┴) to the excitation light. The
In the above equation, t is the time, t is the fluorescence anisotropy (r) is calculated from these
fluorescence lifetime, F0 is the initial fluorescence at measurements by the following equation:
t= 0. Fluorescence lifetimes are measured by either
pulse fluorometry or phase-modulated fluorometry,
both of which are beyond the scope of this chapter. It
may be noted that the fluorescence lifetime is not
directly affected by the energy or intensity of the light
Applications of anisotropy measurements include
emitted. Thus, fluorophores that have similar spectral
determination of equilibrium binding constants for
characteristics can be distinguished if their lifetimes
ligand–receptor interactions, particularly if the
are different. Discrimination between fluorescence
fluorescence of the ligand is monitored.
lifetimes is the basis for the technique known as
fluorescence lifetime imaging microscopy.
C. Fluorescence Instrumentation
4. Quantum Yield Two types of instruments frequently available in life
The quantum yield (f) of a fluorophore is the ratio of science laboratories are fluorescence. Spectrophoto-
the number of photons emitted as fluorescence to the meters have some resemblance to absorption spectro-
number of photons absorbed by the molecule: photometers: both have light sources and accessories to
control the energy of light incident on the sample,
which is typically in a quartz cuvette. In an absorption
spectrophotometer, the detector is in a straight path to
the light source, but in a fluorescence spectrophoto-
meter, the detector is at a right angle to the light source.
Fluorescence spectrophotometers usually have two
The quantum yield can also be expressed in terms of monochromators, hence the wavelength of light that
the fluorescence lifetime: hits the sample and the detector can be modulated
independently. These instruments can collect two types
of spectra: excitation spectra and emission spectra.
Emission spectra are the more common. The sample is
irradiated with light of a particular energy, selected
with the excitation monochromator. The light emitted
from the sample passes through the emission. Mono-
chromator, which is usually scanned over a range
sufficient to collect data from the entire emission band.
General layout of a fluorimeter
Sample
Exc. Mono
To the sample
Detector
Stretching Vibrations
single bond 5 x 105 dyne/cm
double bond 10 x 105 dyne/cm
triple bond 15 x 105 dyne/cm
As the mass of the atoms increases, the vibration
frequency decreases. Using the following
mass values:
C, carbon 12/6.02 x 1023
H, hydrogen 1/6.02 x 1023
Common Applications
• Identification of compounds by matching spectrum of
unknown compound with reference
spectrum (fingerprinting)
• Identification of functional groups in unknown substances
• Identification of reaction components and kinetic studies of
reactions
• Identification of molecular orientation in polymer films
• Detection of molecular impurities or additives present in
amounts of 1% and in some cases as
low as 0.01%
• Identification of polymers, plastics, and resins
• Analysis of formulations such as insecticides and copolymers
Circular Dichroism
The near-UV CD bands of proteins (310–255 nm)
Circular dichroism (CD) is an excellent method for
derive from Trp, Tyr, Phe, and Cys and reflect the
the study of the conformations adopted by proteins
tertiary, and occasionally quaternary, structure of the
and nucleic acids in solution. Although not able to
protein. Although several amino acid side chains
provide the beautifully detailed residue-specific
(notably Tyr, Trp, Phe, His, and Met) absorb light
information available from nuclear magnetic
strongly in the far-UV region of the spectrum (below
resonance (NMR) and X-ray crystallography, CD
250 nm), the most important contributor here is the
measurements have two major advantages: they can
peptide bond (amide chromophore), with n →π* and
be made on small amounts of material in
π→π* transitions at ~220 and ~190 nm, respectively.
physiological buffers and they provide one of the
The far-UV CD bands of proteins reflect the secondary
best methods for monitoring any structural
structure of the protein (α-helix, β-sheet, β-turn, and
alterations that might result from changes in
unordered content). In the case of nucleic acids and
environmental conditions, such as pH, temperature,
oligonucleotides, the aromatic bases are the principal
and ionic strength. This chapter describes the
chromophores, with absorption beginning at around
important basic steps involved in obtaining reliable
300 nm and extending far into the vacuum UV region.
CD spectra: careful instrument and sample
The electronic transitions of the ether and hydroxyl
preparation, the selection of appropriate parameters
groups of the sugars begin at 200 nm, but their
for data collection, and methods for subsequent data
intensity is much weaker than that of the bases, and the
processing. The principal features of protein and
electronic transitions of the phosphate groups begin
nucleic acid CD spectra are then described, and the
further still into the vacuum.
main applications of CD are discussed. These
include: methods for analyzing CD data to estimate
Although CD spectroscopy generally provides only
the secondary structure composition of proteins,
low resolution structural information, it does have two
methods for following the unfolding of proteins as a
major advantages. First, it is extremely sensitive to
function of temperature or added chemical
changes in conformation, whatever their origin, and
denaturants, the study of the effects of mutations on
second, an extremely wide range of solvent conditions
protein structure and stability, and methods for
is accessible to study with relatively small amounts of
studying macromolecule–ligand and macromolecule
material. The principal applications of CD
–macromolecule interactions.
spectroscopy in the study of biomolecules are,
CD, the differential absorption of left- and right-
1. The estimation of protein secondary structure
handed circularly polarized light, is a spectroscopic
content from far-UV CD spectra.
property uniquely sensitive to the conformation of
2. The detection of conformational changes in proteins
molecules, and so has been very widely used in the
and nucleic acids brought about by changes in pH, salt
study of biomolecules. CD often provides important
concentration, and added co-solvents and the structural
information about the function and conformation of
analysis of recombinant native proteins and their
biomolecules that is not directly available from
mutants
more conventional spectroscopic techniques, such
3. Monitoring protein or nucleic acid unfolding brought
as fluorescence and absorbance. The experimentally
about by changes in temperature or by the addition of
measured parameter in CD is the difference in
chemical denaturants (such as urea and guanidine
absorbance for left- and right-handed circularly
hydrochloride)
polarized light, ΔA (= AL − AR). Because CD is an
4. Monitoring protein–ligand, protein–nucleic acid,
absorption phenomenon, the chromophores that
and protein–protein interactions e. Studying (in
contribute to the CD spectrum are exactly the same
favorable cases) the kinetics of macromolecule–
as those contributing to a conventional absorption
macromolecule, macromolecule–ligand interactions
spectrum. In order to show a CD signal, a
(particularly slow dissociation processes), and the
chromophore must be either inherently chiral
kinetics of protein folding reactions. The general
(asymmetric) or must be located in an asymmetric
principles of the most common kinetic methods .
environment. It is the interaction between the
chromophores in the chiral field of the protein that
introduces the perturbations leading to optical
activity.
Instrumentation
CD instruments are commercially available from The CD spectra of membrane proteins are often
several sources: A Peltier system for temperature recorded in detergent solubilized form in order to
control and thermal ramping is an invaluable avoid artifacts arising from differential light scattering
accessory, particularly for studies of the thermal and absorption flattening. The far-UV CD spectra of
unfolding of proteins and nucleic acids. The only proteins (260–178 nm) are intense, and relatively
other significant requirement is for a set of high small amounts of material are required to record them.
quality quartz cuvettes with good far-UV Because all peptide bonds contribute to the observed
transmission with path lengths ranging from 0.1 to spectrum, the amount of material required (measured
10 mm. Cuvettes should always be cleaned in mg/ml) is effectively the same for any protein. The
immediately after use in order to avoid the buildup near-UV CD spectra of proteins are generally more
of hard-to-remove protein deposits. than an order of magnitude weaker than the far-UV
CD spectra. Recording them therefore requires more
Instrument Care and Calibration concentrated material and/or longer optical path
The CD instrument should always be purged with lengths.
high-purity, oxygen-free, nitrogen (generally run at
3–5 L/min) for at least 20 min before starting the Determination of Sample Concentration
light source and throughout the measurements. If Accurate sample concentrations are absolutely
oxygen is present, it may be converted to ozone by essential for the analysis of far-UV CD spectra for
the far-UV light from the high-intensity arc, and secondary structure content and whenever one wishes
ozone will damage the expensive optical surfaces. to make meaningful comparisons between different
Higher nitrogen flow rates will generally be protein or nucleic acid samples. We routinely
necessary for measurements made at very short determine protein and nucleic acid concentrations
wavelengths. The calibration of the instrument using absorption spectroscopy. When the extinction
should be checked periodically. Although several coefficient is known, the concentration can be
CD standards are available, the one used most calculated with considerable accuracy. The absorption
frequently is d10 camphorsulfonic acid (d10-CSA). spectrum should ideally be recorded with temperature
The exact concentration (C) of a solution of d10- control and careful attention should be given to correct
CSA in water (at 2.5 mM) should be determined baseline subtraction, especially when buffers
from an absorption spectrum (using ε285 = 34.5 M-1 containing reducing agents are being used. Highly
cm-1). This measurement also provides a useful scattering samples should always be clarified by low-
check on the wavelength calibration of the speed centrifugation or filtration prior to concentration
instrument, although this can also simply be done determination. If the spectrum still shows significant
by scanning with a holmium oxide filter in the light light scattering, that is, significant background
path and monitoring the voltage on the instrument’s absorption above 315 nm, a correction must be
photomultiplier. applied.
NMR Parameters
Chemical Shift
The chemical shift of a nucleus is the difference
between the resonance frequency of the nucleus and a Nuclear Overhauser Effect (NOE)
standard, relative to the standard. This quantity is The NOE is one of the ways in which the system (a certain
reported in ppm and given the symbol delta, spin) can release energy. Therefore, it is profoundly related
d = (n - nREF) x106 / vREF to relaxation processes. In particular, the NOE is related to
In NMR spectroscopy, this standard is often exchange of energy between two spins that are not scalarly
tetramethylsilane, Si(CH3)4, abbreviated TMS, or 2,2- coupled (JIS = 0), but have dipolar coupling.
dimethyl-2-silapentane-5-sulfonate, DSS, in The NOE is evidenced by enhancement of certain signals in
biomolecular NMR. The good thing is that since it is a the spectrum when the equilibrium (or populations) of other
relative scale, the d for a sample in a 100 MHz magnet nearby are altered. For a two spin system, the energy
(2.35 T) is the same as that obtained in a 600 MHz diagram is as following:
magnet (14.1 T).
Fig. 11: High frequency octopole ion guide for the injection
of ions into an ion trap MS. Compared with a quadrupole
ion guide, it enables a higher precision of guidance