|
Enzymes
are protein molecules that work as natural biological
catalyzers in transforming substrate molecules to
desired products. They are important components of
pharmaceutical, biomedical, and other formulations and
diagnostic assays. Industrial application of enzymes
requires effective techniques for real-time analysis of
enzyme activity and enzymatic reactions. Due to their
complexity, most traditional methods are not suitable
for directly monitoring the enzymatic reactions in real
time in these systems. Therefore, new analytical
approaches for direct measurement of enzymatic reactions
in a broad range of complex media are required. This
paper describes application of novel, high-resolution
ultrasonic spectroscopy (HR-US) for real-time analysis
of the kinetics of enzymatic proteolysis.
Proteolytic
enzymes (proteases), which catalyze the cleavage of
peptide bonds, play an important role in many
physiological processes ranging from blood coagulation
to food digestion. They have a considerable number of
commercial applications and account for a large portion
of worldwide enzyme sales. Proteases are also
extensively used in basic research as a tool for protein
structure determination and sequence elucidation.
Effective characterization of the reactions of enzymes,
including determination of their kinetic parameters,
depends on the availability of experimental techniques
for real-time monitoring of the reactions.
The
most common methods for analysis of kinetics and
activity of proteases are based on UV/visible and
fluorescence spectrophotometric assays. These techniques
require optical activity of either the reactants or
products, or the use of optical markers. They often are
time consuming and require several steps, such as the
precipitation of undigested protein, separation by
centrifugation and pH adjustment, etc. High-Performance
Liquid Chromatography (HPLC), radiolabeling and coupled
enzyme assays have also been developed; however, they
have obvious disadvantages in routine day-to-day
measurements. Therefore, an objective technique that
could be used to continuously monitor a broad range of
enzymatic proteolysis without the need for optical
activity or markers would be an invaluable tool from
both a research and commercial point of view.
This
paper describes application examples of the HR-US 102
ultrasonic spectrometer (Ultrasonic Scientific) to study
the real-time kinetics of enzymatic proteolysis. These
measurements require extremely high resolution and the
ability to work with samples of small volume, which was
a problem for traditional ultrasonic techniques.
High-Resolution Ultrasonic Spectroscopy (HR-US)
overcomes these limitations as well as many of the
drawbacks of its electromagnetic-wave counterparts and
provides a novel, non-destructive method for material
analysis without the need for markers or probes.
HR-US
is based on the measurements of two parameters of
ultrasonic waves, velocity and attenuation. These
parameters are physically independent, allowing access
to different levels of molecular organization.
Ultrasonic velocity is determined by the density and
elastic response of the sample to the oscillating
pressure and so is directly related to the
intermolecular forces and molecular recognition. It is
extremely sensitive to changes in the hydration (solvation)
shell of solute molecules and molecular arrangements in
the analyzed medium. Attenuation is mainly determined by
the energy losses in compressions and decompressions in
ultrasonic waves. The two major factors contributing to
these losses are scattering (followed by attenuation) of
ultrasonic waves in non-homogeneous samples and fast
chemical relaxation. Periodic changes in pressure and
temperature cause periodic shifts in the equilibrium
position of chemical reactions followed by relaxation
back to the equilibrium position, which results in the
energy losses.
|
|
| Fig. 1.
Real-time monitoring of hydrolysis of Z-GLGGA
peptide by Proteinase K using HR-US ultrasonic velocity
measurements (small symbols). The solid lines are
theoretical curves obtained by fitting the experimental
data to the Michaelis-Menton equation. Large squares
present Standard UV spectroscopy data. |
In the
present work, evolution of ultrasonic parameters was
monitored during hydrolysis of two native proteins
(Bovine Serum Albumin [BSA] and casein aggregates from
bovine milk), as well as the hydrolysis of synthetic
pentapeptide, Z-GLGGA (Z is carboxybenzyl), by
Proteinase K (PK). A
complete study of the hydrolysis of native proteins and
protein self-assemblies by a protease requires
monitoring both the molecular process of peptide bond
cleavage and the change in molecule size from a large
aggregated structure to small peptide fragments.
Ultrasonic spectroscopy is capable of real-time
monitoring of both these processes in a single
experiment through changes in velocity and attenuation.
Cleavage of a peptide bond results in substantial
changes in hydration and hence ultrasonic velocity. The
loss of an aggregate or quaternary structure of native
proteins and changes in protein dynamics on hydrolysis
by a protease results in a substantial reduction of the
scattering of the ultrasonic wave and so reduction in
particle size during the process is readily monitored by
changes in attenuation. For comparison, the hydrolysis
of the peptides by PK was independently monitored using
traditional spectroscopic techniques.
|
|
| Fig. 2.
HR-US monitoring of hydrolysis of BSA (1 mg/ml)
by concentrations of Proteinase K; 25 mg/ml (filled
symbols) and 100 mg/ml (closed symbols) and change in
fluorescence over time due to the hydrolysis of labeled
BSA by PK (same concentrations), measured at 515 nm.
Experiments were carried out at 37°C, in 30 mM Tris, pH
8. |
Hydrolysis
of oligopeptide, Z-GLGGA,
by Proteinase K
Figure
1 presents the change in ultrasonic velocity on the
hydrolysis of 1 mg/ml of oligopeptide,
carboxybenzyl-GLGGA (Z-GLGGA), by 100 µg/ml PK in 30 mM
Tris, pH 8, at 37°C. No changes in attenuation were
recorded. Addition of the enzyme (at five minutes)
caused an increase in ultrasonic velocity. The increase
in velocity was followed by saturation in just over one
hour, indicating completion of the reaction. The
percentage of hydrolysis, right scale, was calculated as
a proportion of the current change in velocity to the
total change at saturation level. Also displayed in
Figure 1 is the percentage of hydrolysis of 1 mg/ml Z-GLGGA
by 100 µg/ml PK after 35 and 85 minutes, measured using
the ninhydrin method (squares). The results of the
ultrasonic spectroscopy and UV/ninhydrin experiments
agree very well.
Z-GLGGA
is cleaved by PK to yield the di- and tripeptide
products GL and GGA.
GLGGA +
H2O g
GL + GGA
The cleavage results in a transformation of neutral
amide (C(O)NH) bond and a single bulk water molecule to
a carboxylic acid (COO–) and a charged amine group (NH3+).
R1 –
C(O)NH – R2 + H2O g
R1 – C(O)O–
++NH3 – R2
This
leads to an increase in the hydration, hence a
corresponding increase in ultrasonic velocity for the
hydrolysis of a peptide bond. Fitting the ultrasonic
velocity time profile with Michaelis-Menton equation
(the solid line in Figure 1) allows direct determination
of the kinetic constants of the reaction.
Hydrolysis
of BSA by Proteinase
K
Ultrasonic
profiles of hydrolysis of globular protein BSA (1 mg/ml)
by PK at two different concentrations (25 and 100 µg/ml)
are shown in Figure 2. As already seen for hydrolysis of
the peptides, proteolysis resulted in an increase in
ultrasonic velocity. However, the nature of the change
in the velocity during hydrolysis of high-molecular
weight aggregates or globular proteins is more complex
compared with small oligopeptides.
As the intrinsic compressibility (and density) of
protein globules contributes to the total
compressibility (and density) of the solution the loss
of the protein structure will result in an additional
(to the hydration) increase in ultrasonic velocity.
Figure
2 reveals that increasing the enzyme concentration from
25 to 100 µg/ml did not affect the overall shape of the
time profiles but did result in an increase in the speed
of the change in ultrasonic velocity.
The difference in profiles also indicates that a
deeper level of hydrolysis is achieved by increasing the
concentration of enzyme.
The
insert in Figure 2 presents the measured change in
fluorescence intensity on the hydrolysis of labeled
(self-quenching fluorescence dye) BSA (concentration 1
mg/ml) by PK at 25 and 100 µg/ml. An initial baseline
intensity of 10–12 units (a.u.) was recorded. The
fluorescence increased over time on addition of PK,
similar to the increase in ultrasonic velocity. As
observed in the ultrasonic profiles, increasing the
concentration of PK from 25 to 100 µg/ml resulted in a
deeper level of hydrolysis, reflected in the larger
change in fluorescence intensity.
|
|
| Fig. 3
(A) Ultrasonic
profile of the hydrolysis of casein aggregates (10
mg/ml) by PK (100 mg/ml) measured at four different
frequencies. (B) The change of the aggregates’ radii
over time, calculated from the ultrasonic data using the
particle-sizing module provided with the HR-US 102
spectrometer. |
Hydrolysis
of casein aggregates by Proteinase K and casein particle
sizing
Hydrolysis
of 10 mg/ml casein by 100 µg/ml PK at 20°C, in 30 mM
Tris, pH 8 was monitored at four different frequencies
(5, 7.5, 11, and 13.5 MHz) over 18 hours. The results of
this experiment are displayed in Figure 3A (values
measured at time 0 minutes were subtracted). The
increase in ultrasonic velocity over time due to the
hydrolysis can be attributed to the same factors as in
the case of BSA: the loss of protein aggregated
structure and the hydration of atomic groups.
A
considerable change in the ultrasonic attenuation was
recorded during the hydrolysis of casein by PK in
contrast to the peptide hydrolysis by PK. The
attenuation time profiles are similar (but not the same)
in shape to the velocity plot. A decrease in attenuation
was observed at all frequencies, with higher frequencies
showing a higher magnitude of decrease.
The
overall attenuation of large, colloidal-sized particles
(such as casein aggregate used in this study) dispersed
in a continuous medium is determined primarily by the
intrinsic absorption of the sample components and
scattering of the ultrasound wave on particles. The
scattering level depends on the particle size. As the
size of the protein aggregates decreases during the
hydrolysis process, scattering of the ultrasound wave is
expected to decrease, thus resulting in a reduction of
the excess attenuation. A considerable decrease in
ultrasonic attenuation is indeed observed early on in
the time profile of the hydrolysis of casein by PK.
The
relative changes in aggregate radius over time due to
the hydrolysis by PK were calculated using the
ultrasonic attenuation data and HR-US particle-sizing
software module provided with the HR-US 102
spectrometer; they are plotted in Figure 3B. Before
addition of PK, the average radius of the casein
aggregates was about 130 nm, which should be expected
for casein aggregates. The plot shows that the size of
the aggregates rapidly decreased by over 100 nm during
the first 30 minutes of the hydrolysis, after which the
size of the aggregates continued to decrease over time,
but at a much slower rate.
Conclusion
High-Resolution
Ultrasonic Spectroscopy (HR-US) is a powerful new
technique for real-time monitoring of the enzymatic
proteolysis in different systems. This spectroscopy is
capable of monitoring both the micro structural process
of peptide bond cleavage and the change in molecule size
from a large aggregate structure to small peptide
fragments in a single experiment. Kinetics of the
hydrolysis and the kinetics constants can be determined
directly from the ultrasonic data. The measurements can
be performed in small volumes (down to 30 µL), thus
minimizing the quantities of the samples required for
the analysis. Overall, High-Resolution Ultrasonic
Spectroscopy provides a broad range of new tools for the
analysis of enzymatic proteolysis and other enzymatic
reactions.
For
more information, please contact Ultrasonic Scientific
Ltd. by emailing info@ultrasonic-scientific.com,
visiting www.ultrasonic-scientific.com, or calling +353
1 218 0600.
|
