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New capabilities of high-resolution ultrasonic spectroscopy: Titration
analysis
Article in Spectroscopy -Springfield then Eugene then Duluth- · October 2005
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New Capabilities of HighResolution Ultrasonic
Spectroscopy: Titration Analysis
High-resolution ultrasonic spectroscopy titration analysis is a powerful new tool in research
and analytical laboratory work for quantitative measurements of different processes and
compounds. Here, the authors explore its potential.
Markus Jäger, Udo Kaatze, Evgeny Kudryashov, Breda O'Driscoll, and Vitaly Buckin
T
itration is a common analytical procedure used in modern laboratories. One of the first types of titration was
invented by Joseph Louis Gay-Lussac, known as an
author of “The Law of Combining Volumes.” Today, titration is
an analytical method that allows quantitative determination of
a specific substance (analyte) in a sample by adding to it a second reactant solution of known concentration in carefully
measured amounts until the reaction of definite and known
proportion is completed. To be suitable for a determination,
the end of the titration reaction must be observable. This
means that the reaction should be monitored by an appropriate detection technique, for example, potentiometry, color
indicators, pH, and so forth.
One of the important applications of titration is analysis of
molecular binding. In this case, an appropriate detection system provides quantitative information on the amount of
titrant bound to its target and the nature of the binding. This
allows the measurement of the binding isotherm, which represents the dependence of concentration of bound titrant on the
total or free concentration of the titrant in solution. The binding isotherm then is used to calculate binding constants, stoichiometry, and the free energy of binding. Temperature
dependence of the free energy is used to calculate the entropy
and enthalpy of binding.
The key element of titration analysis is the selection of an
appropriate detector, which can provide quantitative information on the amount of titrant bound or reacted with the analyte. A range of techniques can be employed in titration analysis. The list of these techniques includes potentiometry,
voltametry, electrical conductivity, isothermal titration
calorimetery, UV/Vis absorbance, fluorescence, and others.
However, none of these methods can serve as a universal detector for the binding of titrant to analyte. For example, fluorescence and UV/Vis absorbance require a change in optical
activity of titrant or analyte in the binding as well as optical
transparency of the solution. Therefore, titrations often
require a complex sample preparation procedure such as
24 Spectroscopy 20(10)
October 2005
extraction of analyte to make a solution with required optical
transparency or other procedures such as attaching optical
markers to titrant molecules, etc.
High-resolution ultrasonic spectroscopy (HR-US) is an
analytical technique based upon precision measurements of
parameters (velocity and attenuation) of ultrasonic compression wave propagating through the analyzed sample. This
technique allows direct probing of intermolecular forces and
therefore can be used as a universal detector for titration
analysis. Any change in molecular structure upon the binding
affects intermolecular interactions in the sample and therefore
can be detected with ultrasonic measurements. The measured
ultrasonic titration profile can be recalculated into the binding
isotherm. This technology is extremely sensitive, requires no
markers, and can be used in non-transparent samples such as
cell cultures or dispersions (for example, blood or milk).
Another advantage of the HR-US titration technique is its ability to analyze molecules in their original state without immobilizing procedures or transferring into another environment.
The key factor responsible for “ultrasonic visibility” of
molecular processes is the resolution of the ultrasonic measuring devices, similar to a magnification power of telescopes in
astronomy, which determines the visibility of stars. Novel
principles of ultrasonic detection utilized in high-resolution
ultrasonic spectrometers allow a tremendous increase in the
resolution of ultrasonic instrumentation by several orders of
magnitude when compared with the traditional ultrasonic
techniques. Current applications of this technique include
analysis of chemical reactions, conformational transitions in
polymers and biopolymers, aggregation and gelation phenomena, particle sizing, phase transitions, stability of emulsions
and suspensions, formation of micelles, ligand binding, composition analysis, and many others.
Recently, capabilities of the HR-US technique have been
expanded to titration analysis. HR-US titration spectrometers
allow analysis of most chemical reactions and molecular bindings without or with minimum sample preparation. They do
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Ultrasonic Spectroscopy
not require optical markers and optical transparency and use
only a small volume of sample (down to 0.04 mL).
Experimental
In this example, the Ultrasonic Titration system (Ultrasonic
Scientific, Dublin, Ireland) (Figure 1) was used to analyze the
binding of alamethicin to unilamellar DMPC (1,2-dimyristoyl-3-sn-glycero-phosphocholine) vesicles. Alamethicin is a
peptide antibiotic that forms transmembrane channels and
regulates membrane permeability by acting as a monovalent
cation ionophore. However, while the primary mechanism of
antibacterial activity is not yet known, it is generally agreed
that binding of peptide monomers to the surface of the target
cells causes disruption, permeabilization, or disintegration of
cytoplasmic membranes. It was proposed that depending
upon the conditions, the peptides associate with membranes
in two ways: they either adsorb parallel to the membrane surface (at low peptide/lipid ratios, P/L) or insert perpendicularly into the bilayer (at high peptide ratios), as illustrated in
Figure 2.
Binding of alamethicin depends upon the state of the lipid
membrane, which could be in a fluid (at high temperature) or
gel state (at low temperature). The transition temperature
between the fluid and the gel states for the DMPC membranes
is about 23 °C. In the current example, the binding was analyzed at two temperatures, 17 °C (gel state) and 33 °C (fluid
state).
Figure 1. The HR-US Titration system: Titration module
and HR-US 102 spectrometer are controlled by a PC
using HR-US Titration Analysis software.
The DMPC vesicles in water were prepared by extrusion and
1 mL of 7.54 mM DMPC suspension was loaded into the sample cell of the ultrasonic analyzer. The reference cell was filled
with water. Solution of 5 mM alamethicin in ethanol was used
as a titrant. The titrant solution was injected to the sample
automatically in 1-µL steps. After each injection, the sample
was mixed using double stirring systems that provided effective mixing within seconds. Ultrasonic velocity and attenua-
Change in ultrasonic velocity (m/s)
Adsorption on the membrane
2
1
T = 17 °C
0
-1
-2
T = 33 °C
0.00
0.01
0.02
0.03
Peptide/DMPC molar ratio (P/L)
Insertion into the membrane
and formation of pores
Figure 2. Ultrasonic titration profile of unilamellar vesicles of
DMPC with pore forming peptide alamethicin in water. Change
in ultrasonic velocity in solution caused by addition of the
peptide is plotted as function of the molar ratio of alamethicin
to DMPC (P/L). The curve for 17 °C corresponds to the binding
with the gel membrane and for 33 °C, with the fluid
membrane. (The gel to fluid transition temperature for DMPC
membranes was about 23 °C.)
Circle 22
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Ultrasonic Spectroscopy
200
T = 17 °C
0
-200
-400
0.000
T = 33 °C
0.005
0.010
0.015
0.020
Peptide/DMPC molar ratio (P/L)
Fraction of bound peptide (%)
Figure 3. Differential ultrasonic titration profile of
unilamellar vesicles of DMPC with pore forming peptide
alamethicin in water. The slope of the ultrasonic velocity
curves (change in velocity per unit of P/L ratio) is plotted
as function of the molar ratio of alamethicin to DMPC
(P/L). The curve for 17 °C corresponds to the binding with
the gel membrane and for 33 °C, with the fluid
membrane. (The gel to fluid transition temperature for
DMPC membranes occurs at 23 °C.)
tion in the suspension were monitored over the course of the
titration. The same titration of DMPC vesicles was performed
with just ethanol as titrant to determine the contribution of
ethanol to ultrasonic parameters. The data were subtracted
from the original titration curve and was corrected for dilution
using Titration Analysis software (Ultrasonic Scientific). The
resulting titration curves at two temperatures are given in
Figure 2.
The titration curve at 17 °C (gel state of the membrane)
reveals two major stages of binding. The first stage is observed
at low peptide to lipid (P/L) ratios, up to 0.0026 (or 1 peptide
molecule per 380 lipid molecules). This stage shows a significant increase in ultrasonic velocity and therefore a decrease in
compressibility of the system. This can be explained by
adsorption of alamethicin molecules on the membrane surface. Molecular dynamic simulations (1) show that the helical
structure of alamethicin is stabilized by the hydration water of
the membrane. It is known that the strong binding of hydration water leads to a decrease in the compressibility. The
adsorption of alamethicin molecules on the membrane surface
also can reduce the movements of molecules of lipid, which
will reduce compressibility of the membrane.
At high alamethicin concentration, P/L > 0.0085, the sound
velocity shows a slow linear increase in the velocity with peptide concentration. This stage can be attributed to a solubilization (insertion) of alamethicin in the membranes. The change
in the velocity upon the insertion is less in comparison with
those for the adsorption when calculated per mole of peptide
bound. This is seen clearly in Figure 3, which represents the
slope of ultrasonic titration curve (change of ultrasonic
velocity per unit of P/L ratio). The lower slope of velocity
curve at the second stage can be attributed to a dehydration
of peptide upon the insertion. In addition, the effect of pep-
100
Peptide inserted
into membrane
50
Peptide adsorbed
onto membrane
0
0.000
0.005
0.010
0.015
Peptide/DMPC molar ratio (P/L)
Figure 4. Distribution of bound alamethicin molecules
between the adsorbed onto the surface of DMPC membrane
and the inserted into the membrane states at 17 °C as
calculated from the slope of ultrasonic velocity.
Circle 24
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Ultrasonic Spectroscopy
tide on compressibility of membrane also can be different
compared with the first stage. Assuming that the initial (at P/L
= 0/0.0015) and final (at P/L > 0.01) slopes of the ultrasonic
velocity curve (Figure 2) correspond to the adsorption and
insertion modes of the peptide binding, respectively, the percentage of alamethicin bound to the surface of the membrane
and incorporated in the membrane was calculated and plotted
in Figure 4. Figure 4 allows an estimation of the concentration
corresponding to the beginning of incorporation of alamethicin into the membrane, the critical concentration for insertion or CCI, which is approximately 20 µM (at P/L = 0.0026)
at 17 °C.
At 33 °C (fluid membrane), ultrasonic titration curves
(Figures 2 and 3) show a more complex profile of binding of
alamethicin. The beginning of the binding is significantly different compared with the gel membrane, however, the last
binding stage is the nearly the same for both fluid and gel
membranes. A significant decrease in ultrasonic velocity in a
narrow range of alamethicin concentration (2 m/s within the
P/L range of 0–0.04) is an indication of a cooperative change
in the lipid structure upon the peptide binding. One of possible reasons could be a fusion of lipid vesicles induced by the
peptide. However, further studies of the binding process in a
wider concentration range of lipids and at different temperatures could be essential to clarifying the mechanism of this
process. These analytical tasks can be performed using the HR-
Circle 26
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US technique with expanded titration capabilities.
Conclusion
HR-US titration analysis is a new, powerful tool in research
and analytical laboratory work for quantitative measurements
of different processes and compounds. This technique provides universal detection capabilities for molecular binding, as
any binding affects intermolecular forces in the sample, and
therefore, can be detected with ultrasonic measurements.
Because the measurements do not require any optical transparency, molecular markers or other properties of solution
and solutes, the complex sample preparation procedures in
many cases become obsolete.
Reference
1. P.D. Tieleman, H.J.C. Berendsen, and M.S.P. Sansom, Biophys. J.
76, 3186 (1999). ■
Markus Jäger and Udo Kaatze are with Arbeitsgruppe
Komplexe Fluide, III Physikalisches Institut, Georg-August-Universitaet
Goettingen (Goettingen, Germany).
Evgeny Kudryashov and Breda O’Driscoll are with
Ultrasonic Scientific (Dublin, Ireland). E-Mail info@ultrasonic-scientific.com.
Vitaly Buckin is with the Department of Chemistry at University
College Dublin (Dublin, Ireland).
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