Institute of Pharmacology and Toxicology (Z.Y., Z.D.) and the
Institute of Chemistry (B.C., W.C.), Academia Sinica, Beijing, Peoples
Republic of China
Force spectroscopy between a single acetylcholinesterase (AChE)
molecule and its natural substrates was performed, and the effects of
inhibitors and reactivators on the force spectrum were studied with
atomic force microscopy (AFM). The force spectrum between normal AChE
and its substrates had its special shape. Inhibitors, which inhibit
AChE by occupying the active center of the enzyme, could change the
force spectrum shape noticeably. Reactivators, which reactivate the
inhibited AChE by pulling the inhibitor off the active center of the
enzyme, could make the normal shape of force spectrum reappear. This
meant the shape features of the force spectrum could be used as a good
index to observe the time course of the interactions between a single
AChE molecule and its special inhibitors and reactivators in real time. The results of the real-time observation demonstrated that the inhibition times of soman and sarin on AChE were longer than
2 h and that of eserine, a reversible inhibitor of AChE, was
34 ± 3 min. The reactivation time of HI-6 on soman-inhibited AChE was 6 ± 2 min. These results indicated that AFM was a useful tool in pharmacology and toxicology, and could reveal time information of
the interactions between AChE and its ligands.
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Introduction |
The
principal role of acetylcholinesterase (AChE, acetylcholine hydrolase,
EC 3.1.1.7) is termination of impulse transmission at cholinergic
synapses by rapid hydrolysis of the neurotransmitter acetylcholine
(ACh). In keeping with this requirement, AChE possesses a remarkably
high specific activity, especially for a serine hydrolase, functioning
under bimolecular conditions at a rate approaching the
diffusion-controlled limit. The powerful acute toxicity of organophosphorous compounds is primarily because of their potent inhibition on AChE, and the treatment effects of reactivators on
organophosphorous poisoning are because of their abilities in removing
the inhibitor from the active center of AChE (Ballantyne and Marrs,
1992
). Mild inhibitors of AChE are effective for some human disorders,
such as myasthenia gravis and glaucoma. What is more notable is the
recent research on the effects of AChE inhibitors on Alzheimer's
disease (Krall et al., 1999; Snape et al., 1999). The studies of the
influences of drugs on the interactions between AChE and its natural
substrate ACh are of great interest in pharmacology and toxicology.
The feature of the three-dimensional structure of AChE is a gorge 2 nm
wide, going into half of the enzyme molecule (Sussman et al., 1991).
The catalytic triad is located at the gorge bottom, and the peripheral
choline (Ch)-binding site is at the opening of the gorge (Harel et al.,
1993). More detailed knowledge of the geometry of the active gorge and
the modes of binding of a variety of inhibitors and drugs has been
recently acquired by X-ray diffraction (Boune et al., 1995; Harel et
al., 1995, 1996; Raves et al., 1997; Bartolucci et al., 1999; Kryger et
al., 1999; Millard et al., 1999a,b). Despite all this exciting progress
in studies on AChE, some key features of its enzymatic activity and its
interactions with substrates remain poorly understood
particularly the
single molecular events in the interactions between AChE and its
natural substrate, ACh. The influences of organophosphorous inhibitors
and reactivators on such interactions have not been studied because of
the lack of effective methods. Atomic force microscopy (AFM) (Binig et
al., 1986) has provided such a possibility.
AFM can image not only the biological samples in recognition of
molecules (Smith et al., 1997; Chen et al., 1998; Ret and Fourcad,
1998; Thomson et al., 1999), but also measure the interaction forces
between biomolecules (Florin et al., 1994; Lee et al., 1994; Moy et
al., 1994; Boland and Ratner, 1995; Dammer et al., 1995; Grubmuller et
al., 1996; Stuart and Hlady, 1999). Recently, spatially resolving
single molecular force spectroscopy by AFM has been possible (Heinz and
Hoh, 1999; Müller et al., 1999; Rief et al., 1999a,b) and has
been used in dynamic studies of biomacromolecules (Thomson et al.,
1996; Sagvolden, 1999; Strunz et al., 1999; Zhang et al., 1999a).
Radmacher et al. (1994) observed the activity of lysozyme through
measuring the configuration changes of the enzyme by AFM. In our
previous work, we studied the intermolecular forces between
acetylcholine and acetylcholinesterases (Zhang et al., 1999b). The
basis of the AFM measurement of the interactions between biomolecules
is the recording of the force spectrum, which is a plot of the
interactive force between biomolecules as a function of the separation
between them. A force spectrum is like a fingerprint that identifies a
particular physical system (Jarvis and Tokumoto, 1997).
Characteristics of a force spectrum are dependent on the nature of the
biomolecules and the bonding potential between them. The main purpose
of the present study is to study the characteristics of the force
spectrum between a single AChE molecule and its substrate, ACh, and the
influences of inhibitors and reactivators on it.
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Experimental Procedures |
Materials and Instruments.
Torpedo AChE was purified by
methods described in the literature (Sun et al., 1985). ACh, Ch,
eserine, mercaptopropionic acid (MPA), mercaptoacetic acid (MAA), and
N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide
(EDC) were purchased from Sigma Chemical Company (St. Louis, MO).
(1-(((4-Aminocarbonyl)pyridino)methoxy)methyl)-2-(hydroxy-imino) methyl)pyridinium dichloride monohydrate (HI-6), pralidoxime-2-chloride (2-PAM), pinacolyl methylphosphonofluoridate (soman), isopropyl methylphosphonofluoridate (sarin), and atropine were provided by the
Beijing Institute of Pharmacology and Toxicology (Beijing, China).
NanoScope, an atomic force microscope, fluid cell, and the
Si3N4 tip of a scanning
probe microscope, which had a cantilever of 200 µm and a spring
constant of 0.12 N/m, were products of Digital Instruments (Santa
Barbara, CA). Gold-plated mica was obtained from Molecular Imaging
(Phoenix, AZ).
Immobilization of AChE on Substrate.
Immobilization of AChE
was performed according to the method described by Legget et al.
(1993). First, gold-plated mica (GPM) was immersed into
10
2 mol · l1
solution of MPA for 30 min to allow the S-Au bond to form between the
gold and the hydrosulfide group of MPA. Next, GPM was immersed into
102 mol · l1
solution of EDC for 1 h to activate the carboxyl of MPA. Then, GPM
was immersed in 1.2 mg · ml1 AChE
solution for 12 h to allow a peptide bond to form between the
carboxyl of MPA and the free amino group of basic amino acids of AChE
(Fig. 1A, bottom). Finally, GPM was
removed and washed in flowing triple-distilled water for 30 s to
remove the noncovalently absorbed AChE molecules. AChE immobilized in
this way may withstand scanning of the tip. In recording force
spectrum, the depression of the tip on one AChE molecule may denature
it, and the normal force spectrum could not develop any more. At this
time, we would align the tip to another AChE molecule. More than 300 force spectra might be recorded in one AChE molecule.
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Fig. 1.
Schematic illustration of the immobilization of AChE
on AFM substrate and ACh on the tip (A) and the recording of force
spectrum (B). For the immobilization of AChE on the substrate, a gold
film was evaporated onto the fresh mica surface, and AChE was
immobilized onto the gold film through MPA (A, bottom). For the
immobilization of ACh, the Si3N4 tip was also
plated with gold. ACh was linked to the gold film by sulfur atoms (A,
top). The tip was installed in the tip frame of the fluid cell, which
was placed on the surface of the substrate (B). Force spectrum was
recorded and observed in real time through the computer screen when the
ACh-modified tip repeatedly approached and retracted from an AChE
molecule.
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Linking of ACh to the Tip Surface.
A gold film was
evaporated onto the tip surface (Boland and Ratner, 1995). The
gold-plated tip was immersed in 102 mol
· l1 MAA for 30 min to allow the S-Au bond
to form between gold and the hydrosulfide group of MAA. Then, the tip
was immersed in 102 mol · l1 Ch solution for 48 h to allow the ester
bond to form between the carboxyl of MAA and the hydroxyl of Ch.
Consequently, ACh formed on the gold film through a sulfur atom (Fig.
1A, top). One modified tip may be worn out after the force spectroscopy of 10 to 20 AChE molecules; therefore, a tip would be changed after a
force spectroscopy in the experiment of 10 AChE molecules.
Force Spectroscopy.
An AFM tip was installed in the tip
frame of the fluid cell, and GPM was mounted onto the top of the
scanner (Fig. 1B). The fluid cell was placed on the surface of GPM and
filled with 50 mmol · l1
phosphate-buffered solution (PBS) (pH 7.4), and the force spectroscopy was conducted at a temperature of 25°C. First, AChE was imaged by
horizontal scanning of the tip on the surface of the GPM. After a clear
image of AChE was obtained, one of the AChE molecules was selected as
the center of the scanning, and the scanning range was reduced to the
size of the AChE molecule. Thus, the tip was aligned to one of the AChE
molecules. Then the scanning mode was changed into force mode in which
the tip repeatedly approached and retracted from AChE. One force
spectrum was recorded every time the tip with ACh approached and
retracted from AChE. By comparing the shape of force spectrum, it could
be ensured that the tip had been aligned to AChE, because the force
spectrum between ACh and AChE was quite different from that between the
tip and the blank areas. To estimate the influence of the tip
materials, force spectrum between AChE and the unmodified tip was also
recorded for control.
Observation of the Effects of Inhibitors and Reactivators on the
Force Spectrum between ACh and AChE.
The force spectrum between
ACh and normal AChE was first recorded and observed. Then, GPM with
AChE was removed from the scanner and immersed into inhibitor solutions
for 30 min for AChE to be inhibited. The GPM with inhibited AChE was
remounted onto the scanner, and the force spectrum between ACh and
inhibited AChE was recorded and observed once more. After the force
spectrum between ACh and inhibited AChE was recorded, the GPM with
inhibited AChE was immersed into reactivator solutions for 30 min for
inhibited AChE to be reactivated and the force spectrum between ACh and reactivated AChE was recorded for the third time. The development rates
of the force spectrums with special shape features were expressed as
the percentage in all of the studied AChE molecules. The force spectrum
between ACh and AChE was identified by observing the relation of the
concentrations of special inhibitors and reactivators with the
development rate of the characterized force spectrum.
Real-Time Observation of the Effects of Inhibitors and
Reactivators on Force Spectrum Shape.
The effects of inhibitors
and reactivators could be observed in real time. In such observations,
GPM with AChE was not removed from the top of the scanner. Solutions of
inhibitors or reactivators may be injected into the fluid cell through
the fluid-in tube at a desired time to continually observe the changes
in the shape features of force spectrum, whereas the tip was kept
aligned to the same AChE molecule. After the shape features of the
normal force spectrum were confirmed, solutions of inhibitors were
injected and the time for the features of normal force spectrum to
disappear was recorded. Reactivators were given regularly after the
disappearance of the features of the normal force spectrum, and the
time for the normal force spectrum to recover was recorded. If the
shape of force spectrum was changed by injection, the experiment was discontinued. Only when the force spectrum shape was not influenced by
injection did the experiment continue to observe the changes in force
spectrum shape. Each experiment was performed in a single enzyme
molecule and observed for 1 h for inhibition or reactivation. The
results were statistically calculated from all studied molecules from
different enzyme preparations.
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Results |
AFM Image of AChE.
AChE was dispersed ellipsoid particles
under AFM (Fig. 2). There were broad
blank areas around AChE molecules, which made it easy to select a
single AChE molecule or the blank area as the center of scanning to
record force spectrum between ACh and AChE or that between tip and
blank areas.
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Fig. 2.
AFM image of AChE. Contact mode in PBS. AChE was
dispersed ellipsoid particles under AFM. There were broad blank areas
around AChE, in which there was no AChE. Scan size, 300 × 300 nm.
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Shape Features of Force Spectrum between ACh and Normal AChE.
The force spectrum between ACh and normal AChE had the same shape as in
Fig. 3A. During the approaching of ACh to
AChE, when the distance between them was larger than the range of
interaction force, the force spectrum was kept in a horizontal line
(Fig. 3A, 
). As ACh was getting into the range of attractive
interactive force, a downward force peak of 220 ± 20 pN (100 AChE
molecules from 25 samples; the following statistical data were the same as this) appeared (Fig. 3A, 
). With ACh getting closer to AChE, a
repulsive force developed between them and force spectrum turned upward
(Fig. 3A, 
). During the process of ACh retracting from AChE, a
reduction of repulsion was first observed and then a large adhesive
force developed between them (Fig. 3A, 
), which had a value of
750 ± 35 pN. When separation reached a certain level, the
adhesion force first quickly decreased (Fig. 3A, 
) to 450 ± 24 pN and then there was a slower adhesion decrease (Fig. 3A, 
) to
280 ± 21 pN. At last the adhesion once more quickly decreased to
the horizontal line (Fig. 3A, 
). As an entire normal force spectrum,
it had the following two features: an attraction peak in an approaching
line (Fig. 3A, ) and the fast-slow-fast decay of the adhesion (Fig.
3A, , , ) in the retracting line. In all of the force spectra
between ACh and normal AChE, 86 ± 10% had the same shape as in
Fig. 3A, which developed only when the ACh-modified tip was aligned to
AChE, and the remainder had the same shape as in Fig. 3C. Between ACh
and the blank areas or between the unmodified tips and AChE, all of the
force spectra had the same shape as in Fig. 3C. These results indicated
the force spectrum in Fig. 3A specifically developed between ACh and
normal AChE, whereas that in Fig. 3C was nonspecific.
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Fig. 3.
The real force spectrum recorded by atomic force
microscopy (left) and the schematic illustration of the characteristics
of force spectrum (right) between ACh and AChE. A, NFS that had two
characteristics: the attraction peak (piece ) in the approaching
line and the curve composed of pieces , , and in the
retracting line. B, force spectrum between ACh and soman-inhibited
AChE, which was obviously different from A in shape. Attenuation of the
adhesion was a curve composed of pieces , , and in A, but it
was a straight line in B. C, force spectrum between ACh and
eserine-inhibited AChE, which was also obviously different from NFS in
shape. In C, the attraction peak in the approaching line seemed to
disappear in addition to the changes in the retracting line.
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Influences of Special Inhibitors on the Shape of Force Spectrum
between AChE and ACh.
The normal force spectrum (NFS) as in Fig.
3A could never be seen after AChE had been treated in
108 mol · l1
soman solution, which inhibited AChE by occupying the active center of
AChE (Ballantyne and Marrs, 1992). In all of the force spectrums
between ACh and soman-inhibited AChE, 84% ± 5 had the shape as in
Fig. 3B, which was obviously different from that in NFS and Fig. 3C in
shape. The decaying of adhesion in Fig. 3B, , was a straight line
rather than a curve as in NFS. The adhesion in Fig. 3B had a value of
620 ± 32 pN, smaller than that in NFS (p < 0.01). Sarin, which was another special inhibitor of AChE occupying the
active center of the enzyme (Ballantyne and Marrs, 1992), had the same
effects on force spectrum shape as soman. In all of the force spectrums
between sarin-inhibited AChE and ACh, the development rate of the force
spectrum as in Fig. 3B was 85 ± 8%. The influence of eserine, a
reversible inhibitor with a positively charged quaternary ammonium
group in molecules, on the force spectrum shape was different from that
of soman and sarin. The force spectrum in Fig. 3C was recorded between
ACh and eserine-inhibited AChE, which was nonspecific. The attraction peak in the approaching line of the force spectrum between ACh and
eserine-treated AChE had disappeared in addition to the changes in
retracting line (Fig. 3C). The development rate of NFS decreased with
the concentration of inhibitors. Linear regression revealed high
negative correlation coefficient (p < 0.01) between
development rate of NFS and the concentration of inhibitors (Fig.
4). It may be seen from Fig. 4 that soman
had no effect on the development of the normal force spectrum at a
concentration of 1012 mol · l1 and decreased it to zero, with
108 mol · l1
having the strongest effect. Sarin at 1011
mol · l1 had no effect and at
107 mol · l1
decreased it to zero, thus having the effect weaker than that of soman.
Eserine at 1010 mol · l1 still had no effect and did not decrease the
development rate to zero even at the concentration of
106 mol · l1
having the weakest effect. Atropine, a drug that did not occupy the
active center of AChE, could not decrease the development rate of NFS.
These results clearly demonstrated that the attraction was related to
the electrostatic interaction between ACh and AChE, the adhesion
decaying curve of NFS was related to the active center of AChE, and the
entering of ACh into the active center was necessary for the formation
of the adhesion curve of NFS. The inhibitors occupying the active
center of the enzyme made the adhesion curve change into a straight
line.
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Fig. 4.
Relation between the development of NFS after 30 min
of inhibitor treatment and the concentration of inhibitors.  , soman;
 , sarin;  , eserine. Sample numbers are 10 to 20 for every
inhibitor. p < 0.01 for the three Rs.
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Effects of Reactivators on the Shape Features between ACh and
Inhibited AChE.
After soman-inhibited AChE was reactivated with
effective reactivator HI-6, which could pull soman from the active
center of AChE, the force spectrum regained its normal shape as in Fig. 3A, namely the so-called NFS. HI-6 also made NFS reappear between ACh
and the sarin-inhibited AChE. 2-PAM, another reactivator of AChE, could
make NFS reappear between ACh and sarin-inhibited AChE, but could not
make NFS reappear between ACh and soman-inhibited AChE. Atropine, a
drug that had no reactivative effects, had no influences on force
spectrum shape. As expressed in Fig. 5,
the development rate of NFS was directly proportional to the
concentration of the reactivator (p < 0.01). The
effect of HI-6 on soman-inhibited AChE was weaker than that on
sarin-inhibited AChE. 2-PAM had an effect on sarin-inhibited AChE,
whereas there was no obvious effect on soman-inhibited AChE. These
results were consistent with early pharmacological research (Ballantyne
and Marrs, 1992) and revealed further that the adhesion curve of NFS
was related to the active center of AChE. The entrance of ACh into the
active center of AChE was an indispensable requisite for the formation
of the adhesion curve.
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Fig. 5.
Relation between the development of NFS after the
inhibited AChE was treated with reactivators for 30 min and the
concentration of reactivators. , HI-6 on soman-inhibited AChE; ,
HI-6 on sarin inhibited AChE;  , 2-PAM on soman-inhibited AChE;  ,
2-PAM on sarin-inhibited AChE. p < 0.01 for HI-6
on soman- and sarin-inhibited AChE and 2-PAM on sarin-inhibited AChE.
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Real-Time Monitoring of the Effects of Inhibitors and
Reactivators.
The findings of the shape features of the force
spectrum between ACh and AChE made it possible to monitor the
interactions between the enzyme and its inhibitors and reactivators at
the single enzyme molecule in real-time through a computer screen. In
Fig. 6, three force spectra were recorded
in each of the AChE molecules. Fig. 6A shows the appearance of NFS.
Three minutes after the injection of soman solution, NFS disappeared
(Fig. 6B). This period was preinhibition time, representing the time
for soman to diffuse into and occupy the enzyme's active center. Ten minutes after the injection of HI-6 solution, the special features of
NFS developed again (Fig. 6C), thus indicating that the reactivators had pulled soman off the enzyme. This period was reactivation time,
representing the time for reactivators to pull the inhibitor off the
enzyme. In this experiment, we made 100 observations on 100 AChE
molecules (from 13 samples). On all AChE molecules, NFS shape features
never disappeared after PBS was injected, and inevitably disappeared
after the solution of soman was injected and reappeared on 49 molecules
after the solution of HI-6 was injected; it never did so after PBS was
injected. The preinhibition time and reactivation time were,
respectively, 5 ± 3 min and 6 ± 2 min for these AChE molecules. For reversible inhibitors, such as eserine, we could observe
the time of the inhibitor staying in the enzyme. Fig. 6, D to F, were
force spectra recorded in one AChE molecule, showing the influences of
eserine on force spectra. Preinhibition time for eserine was 12 ± 3 min. The reactivation time was 34 ± 3 min, representing the
inhibition time on AChE, which meant eserine stayed in the active
center of AChE for 34 ± 3 min and finally left the enzyme through
the interactions between the enzyme and the inhibitor.
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Fig. 6.
Real-time monitoring of the interactions between
enzyme, inhibitors, and reactivators. A, B, and C were the force
spectra recorded in one AChE molecule. A, NFS, the attenuation curve
changed into a straight line (B) 3 min after the injection of soman and
recovered its curve (C) in 10 min after injection of reactivator HI-6.
D, E, and F were the force spectra recorded in another AChE molecule.
D, NFS. Normal shaped features disappeared (E) 5 min after the
injection of eserine and automatically recovered (F) in 30 min.
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Discussion |
The results of our experiment undoubtedly demonstrated the shape
features of the force spectrum between ACh and AChE through nonreal-time and real-time experiments. The results of nonreal-time experiments resulted in the distinguishing of ACh-AChE force spectrum from that between ACh and the impurity proteins through statistically observing the influences of special AChE inhibitors and reactivators on
the force spectrum. Real-time experiments took single AChE molecules as
the object of study and observed the force spectrum changes caused by
drugs in the same AChE molecule. The results of real-time observation
were more reliable. Special inhibitors could change the shape features
of the force spectrum by occupying the active center of AChE.
Reactivators could make the normal features of the force spectrum
reappear by removing the inhibitor from the active center of the
enzyme. The significance of this finding was that the AFM spectroscopy
between ACh and AChE could be applied to many aspects of the life
sciencessuch as enzymology, toxicology, and pharmacologyas a
powerful tool to study the interactions between AChE and its
substrates, inhibitors, and/or reactivators at a single molecule in
real time, which was not achievable by other present biological techniques.
ACh bore one positive charge and AChE had eight negative charges
(Ripoll et al., 1993). Our recent work demonstrated the attraction peak
in the attracting line was developed from the electrostatic interactions between ACh and AChE (Zhang et al., 1999b). The present results that the inhibitors without positive charges did not influence the attraction, whereas the inhibitors with positive charge made the
attraction disappear, also indicated that the attraction developed from
the electrostatic interaction. An explanation for the formation of the
adhesion curves was complex. The results that inhibitors changed
attenuation curves of the adhesion into straight lines and reactivators
made the straight lines change back into curves indicated the
attenuation curve was related to the active center of AChE. Based on
the molecular structure of ACh and AChE, the mechanism for the
formation of the adhesion curve may be explained with the aid of Fig.
7. When the tip with ACh moved closer to the active gorge of AChE, some ACh molecules could then get into the
gorge; the remainder became blocked at the top of the gorge (Fig. 7B).
Now, the ACh molecules, which moved into the gorge, still did not
arrive at the bottom of the gorge because ACh molecules (0.9 nm;
calculated from bond length) were shorter than the depth of the gorge
(2 nm; Sussman et al., 1991). With further downward movement of the
tip, AChE was depressed by the repulsion and the ACh molecules that
entered the gorge came into contact with the bottom of the gorge (Fig.
7C). The two-part ACh molecules formed adhesive interactions separately
with the top surface of AChE and the bottom of the active gorge. During
the process of ACh leaving AChE, the adhesive interaction between ACh
and the bottom was stretched and ruptured (Fig. 7D). The rupture of the
bottom adhesive interactions caused the rapid decrease of the adhesion, forming the first fast attenuation piece of the adhesion curve (Fig.
3A, ). After the rupture of the bottom adhesion, AChE was stretched
by the top adhesion and recovered from its depressed status (Fig. 7E).
In this phase, there was no rupture of intermolecular adhesive
interactions, and variation of the force only depended on the
electrostatic interactions and the interior adhesion of the AChE
molecule so that the attenuation of the adhesion was slower, forming
the slow part of the adhesion attenuation (Fig. 3A, ). With the tip
moved further away from AChE, the adhesion between ACh and the top of
the active gorge of AChE ruptured at last, and the adhesion once more
rapidly decreased to the horizontal line, forming the second fast piece
of the adhesion (Fig. 3A, ). Although further studies are required,
the influences of inhibitors and reactivators provided rather strong
evidence for these speculations. At least it may be affirmed that the
special features of the force spectrum between ACh and AChE are related
to the electrical properties and the spatial structures of AChE and
ACh. Inhibitor molecules impaired the entering of ACh into the active
center by occupying the active center or changing the spatial structure
of AChE by the interactions between it and AChE so that the shape of
the force spectrum was changed. Reactivators could pull inhibitors out
of the active center of AChE so that it could make normal force
spectrum develop again.
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Fig. 7.
Schematic illustration of the ACh-AChE relative
position in the recording of the force spectrum to explain the
formation of the adhesion curves. A, ACh-AChE position relation before
contact. When they reached the range of attractive interactions, an
attraction peak developed. AChE had a gorge leading to the active
center, which was called the active gorge. B, ACh contacted with AChE.
Part of the ACh molecules entered the active gorge, and the remainder
were blocked at the mouth of the gorge and contacted with the top of
AChE. C, the tip moved further toward AChE, and repulsion developed
between ACh and AChE. AChE was depressed and the ACh that entered the
active gorge reached the bottom of the gorge. D, the tip retracted from
AChE; the adhesive interactions between ACh and the bottom of the gorge
ruptured first. E, AChE recovered its form from the depressed status.
F, adhesive interactions between ACh and the top of AChE ruptured
finally.
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As an example of the application of AFM, the time curse of the
interactions between AChE and its special inhibitors and reactivators was studied. Through this experiment, we could get information about a
chemical. How long does it stay in the active center of the enzyme? How
fast does a reactivator reactivate the enzyme? The significance of
preinhibition time and reactivation time was that we could estimate the
speed of the development of the effects of an inhibitor on the enzyme
from preinhibition time by estimating the permanency of the inhibiting
effects of inhibitors from inhibition time and reactivating potency of
reactivators from reactivation time. These indexes were of importance
in molecular pharmacology, toxicology, and enzymology.
The real-time monitoring of drug effects included two key steps. The
first was to align the tip to one AChE molecule and find a typical
normal force spectrum. Then, the solutions of inhibitors were injected
into the fluid cell to observe the shape changes of force spectrum, and
the reactivator solutions were injected to observe the changes in the
spectrum while the tip was kept aligned to the same AChE molecule.
Although it was difficult to keep the tip aligned to the same AChE
molecule when the solutions were injected, it was possible if the
operation was careful enough. In fact, this difficulty was relatively
less important because the tip and AChE had considerable size. In
addition, the changes caused by operation of the injection could be
distinguished from that produced by inhibitors by the times when NFS
disappeared. The former when or immediately after injection, but the
latter was at least several minutes after the injection. The experiment was continued only in the case that the shape feature of force spectrum
had not been changed by injection.
In summary, we found a way to record the force spectrum between a
single AChE molecule and its natural substrate, ACh. The force spectrum
between a normal AChE and ACh had its special shape features, including
the attraction peak in the approaching line and the curve-like decaying
of the adhesion. These shape features disappeared when the enzyme was
inhibited by its special inhibitors and redeveloped when the inhibitors
were pulled off the active center of the enzyme by reactivators. The
interactions between enzyme molecules and inhibitors or reactivators
could be monitored with force spectroscopy in real time at a single
molecule level, just like the heart function of a man could be
monitored with electrocardiography. This method can be used in other
enzyme-ligand systems by studying the shape features of force spectrum.
It is believed that AFM will become a useful tool for life science research.
AChE, acetylcholinesterase;
ACh, acetylcholine;
AFM, atomic force microscopy;
Ch, choline;
MPA, mercaptopropionic acid;
MAA, mercaptoacetic acid;
EDC, N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide;
HI-6, (1-(((4-aminocarbonyl)pyridino)methoxy)methyl)-2-(hydroxy-imino)methyl)pyridinium
dichloride monohydrate;
2-PAM, pralidoxime-2-chloride;
soman, pinacolyl
methylphosphonofluoridate;
sarin, isopropyl methylphosphonofluoridate;
GPM, gold-plated mica;
PBS, phosphate-buffered solution;
NFS, normal
force spectrum.
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Abstract
Introduction
)-huperzine A.
Nat Struct Biol
4:
57-63[Medline].
