The a researchers thought that even whenever our brain

The instant venture of
the aesthetics and the neurosciences is to find out the reason of alertness or
somewhat how the material brain produces our in Material sense of awareness.
Some scientist says that consciousness is an operation of brain, but as consciousness
can happen when brain is not working that’s why some scientists thinks that
consciousness is not physically related to body or brain. As the brain is a
material object, consciousness can understand 
by study of science. The human brain has unbelievable abilities and it’s
a complicated mass of tissue. An 
important part in learning and remembering is played by microtubules as
they focus on neurotransmitter function. Microtubules could connect the memory
and consciousness because these two processes are related to each other. The
total brain protein consists of 15% tubulin, tubulin is protein which
polymerize and arrange in hexagonal cylinders to form microtubules. The
heterodimer tubulin subunit’s shape straight or curved tells the arrangements
of microtubules polymers . As the process by which brain functions to cause
consciousness is not known that’s why the process by which consciousness is
limited by anaesthetics is not well explained. Tubulins have the pi
electron-rich indole rings, has the distance of 2mn,  on the smaller non-polar regions . Penrose-
Hameroff Orchestrated Objective Reduction (Orch-OR) Theory explains that
entangling of  these electrons occurs
because these are close enough.The arrangement of tubulin in presence of
anesthetics is explained in this paper.

Introduction

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Consciousness could be
summarized in many aspects. A number of a researchers thought that even whenever
our brain is not working the consciousness is continued. It can be explained in
other words that the consciousness is a term that is aparted from physical body
and the brain. From scientific point of view consciousness is brain
functionality. Brain is an object or material for the study of science. The
brain of the human is a very complicated mass tissue granted with the
outstanding abilities.in higher animals like humans the brain is a centre for
controlling the CNS.  In these days
several scientists as well as researchers are busy in decoding the secret of the
consciousness. Now here a question arises that can a strong brain imaging
technology define consciousness that is an immaterial thing in the nature. the
consciousness theory explained by Penrose and Hameroff, consciousness arises
from brain and target specially on the synapses complicated computation which
will let the communication within the neurons. The Neurons contain a
well-organized pattern of the microtubules due to a type of proteins high in
number called special nonmotor microtubule (MAPs) attacted with the nerve cells.
It was said that the many organizations of MT between the dendrites and the
axons can perform a special function in signaling of neurons. Evidences shows
that a connection exists within the performance of MT and the imaginary
functions by explaining that within the interval when the accumulation of the
synapses and also the imaged information happens at the  high degree the  cortex (visual) of brain yields a very high
number of the tubulin 1.  A latest
connection within the microtubules and the cognition has studied having the patients
of the Alzheimer’s disease 2.

Materials and Methods

Tubulin (#TL238), taxol
(#TXD01), GTP (#BST06) and General Tubulin Buffer (#BST01) (Microtubule/Tubulin
Biochem kit Cat#BK015) are supplied by Cytoskeleton Inc. Denver, CO. USA.

Preparation of buffer T

GTP stock solution (100
mM) is added to General Tubulin Buffer (80 mM PIPES pH 6.9, 2 mM MgCl2, 0.5 mM
EGTA) at a final concentration of 1 mM GTP. The buffer T will be stable for 2-4
hours on ice.

Fluorescent reporter
buffer

Fluorescent reporter
buffer contains 80 mM Piperazine-N,N’-bis{2- ethanesulfonic acid} seuisodium
salt; 2.0 mM Magnesium chloride; 0.5 mM Ethylene glycol-bis (b-amino-ethyl
ether) N,N,N’,N’-tetra-acetic acid, pH 6.9, 10 ?M DAPI
(4′,6-diamidino-2-phenyindole).

Tubulin Reconstitution

1 mg of lyophilized
tubulin is resuspended in 1 ml of buffer T at 0-4°C (final tubulin
concentration is 1 mg/mL). The reconstituted tubulin solution is not stable and
needs to be used soon after its preparation. Propofol (2,6-di-isopropylphenol)
(Anesthetic) is supplied by MP Biomedicals, Mumbai. Three different
concentrations of Propofol (0.5, 1.0 and 100 mM) were prepared for the study.
Throughout the experiments, all solutions were prepared in MilliQ water. pH
measurements were carried out using EUTECH instruments pH 510.

Kinetics study of
polymerization of bovine brain tubulin in presence of propofol

Polymerization of
Bovine brain tubulin was studied with spectrophotometric technique. The
kinetics of microtubule assembly (bovine brain tubulin) was monitored by Eon
Spectrophotometer (Biotek) at 350 nm for 1-2 hours with the time interval of 1
minute at 37°C. Polymerization of tubulin protein is carried out using standard
protocol 4-8. The effect of Propofol on polymerization of tubulin was also
studied. Chemically, propofol is 2,6-di-isopropylphenol. It has 95-99% protein
binding affinity. Its half-life is 30-60 minutes. Propofol has been proposed to
have several mechanisms of action, both through potentiation of GABA receptor
activity, thereby slowing the channelclosing time, and also acting as a sodium
channel blocker. EEG research upon those undergoing general anesthesia with
propofol finds that it causes a prominent reduction in the brain’s information
integration capacity at gamma wave band frequencies. So the effect of different
concentrations of propofol (0.5, 1.0 and 100 mM) on polymerization of tubulin
in presence of guanosine tri-phosphate (GTP) was also monitored. As Tubulin
proteins do not polymerize into microtubules in the presence of zinc ions and
guanosine di-phosphate (GDP), an attempt was also made to understand the exact
mechanism of propofol binding to tubulin in presence of zinc ions and guanosine
di-phosphate using different concentrations of propofol (0.5, 1.0 and 100 mM).

Circular Dichroism (CD)
Measurements

The isothermal studies
of Tubulin by CD measurements were carried out with Chirascan, a polarimeter of
Applied Photophysics equipped with a Quantum Northwest-TC125, a Peltier-type
temperature controller. The instrument was calibrated with
d-10-camphorsulfonicacid. All the isothermal CD measurements were made at 25°C.
Spectra were collected with 20 nm/min scan speed, 0.1 nm data pitch, and a
response time of 2 s. Each spectrum was the average of 10 scans. The Far-UV CD
spectra (200–260 nm) were taken at protein concentrations of 0.889 mg/ml in a
cell of 0.1 cm path length. All spectra were smoothed by the Savitzky-Golay
method with 25 convolution width. CD values (?) in mdeg, were obtained from the
instrument readings.

TRFS measurements

Excited-state lifetime
measurements were performed using a timecorrelated single photon counting
(TCSPC) spectrometer (Edinburgh FLS920). For our experiments a LASER having its
central wavelength at 375 nm was used as the source for exciting the DAPI present
in fluorescent buffer. Emission was subsequently collected at 440 nm through a
single monochromator with a 5 nm bandpass over a total time range (TAC) of 100
ns for all samples. Emission polarizer was set at 55.4 degree magic angle to
exclude rotational anisotropy lifetime decay to simple decay life time data.
Emission decays were fit with appropriate instrument response functions (IRF)
collected using a scattering solution. The FWHM (full width at half-maximum) of
the IRFs collected was typically in the range of ~120 ps.

Results and Discussion

The kinetics of
microtubule assembly (bovine brain tubulin) was monitored by Eon
Spectrophotometer (Biotek) at 350 nm for 1-2 hours with the time interval of 1
minute at 37°C. Polymerization of bovine brain tubulin was carried out with and
without guanosine triphosphate (GTP) (Figure 1). Bovine brain tubulin in
presence of GTP showed different polymerization- depolymerization behaviour
when compared to that without GTP (Figure 2). In presence of GTP, the
microtubule polymerization was found to be stable to some extent and less
dynamic. In absence of GTP, the microtubule was found to be less stable. This
also showed that GTP, not only enhances the rate of polymerization, but is
essentially required for polymerization. The effect of taxol was also seen on
polymerization of microtubule in presence of GTP and glycerol buffer. It shows
that microtubules are stabilized in presence of taxol (Figure 3). The effect of
different concentrations of propofol (0.5, 1.0 and 100 mM) was also seen on
polymerization of tubulin in presence of 1 mM GTP and glycerol buffer (Figures
4-9). Figure 1 is the control for Figures 4-6. It was seen that propofol
affected polymerization or self organization behaviour with all concentrations
of propofol. With 0.5 mM propofol, polymerization was affected upto 10 minutes,
with 1 mM Propofol, the rate of polymerization was affected upto 30 minutes and
with 100 mM propofol, polymerization was affected upto 60 minutes. When the
effect of propofol was seen on tubulin polymerization in the presence of GDP
and zinc ions, it was observed that polymerization was strongly affected with
all three concentrations of propofol and the same trend was observed with all
three sets of experiments. With low concentration of propofol (0.5 mM), the
polymerization or self-organizational behaviour of tubulin was affected upto 10
minutes (Figure 4). From this, it is inferred that the effect of propofol
diminishes after 10 minutes with 0.5 mM Propofol as its half-life is 30- 60 minutes.
But with a little higher concentration of propofol (1 mM), self-organizational
behaviour was affected upto 30 minutes (Figure 5). When polymerization of
tubulin was studied with 100 mM propofol, the self-organizational behaviour of
microtubule was strongly affected upto one hour (Figure 6). From above
observations, it is inferred that propofol (anesthetic) binds to hydrophobic
pockets of tubulin via weak Vander waals london dispersive forces 9.
Propofol’s effect is time- and dose-dependent, and can be reversed when
propofol is removed. For further experiments, we have chosen propofol’s dose to
be 100 mM 10-20. As zinc chloride was added to tubulin protein, a notable
rearrangement of spectrum occurred with lowering in major minima as well as
slight changes in the shape of spectra. A lowering in negative value of
ellipticity at 208 nm and 222 nm indicated a sign of ?-helical reduction due to
intramolecular H-bonding rearrangement. After the addition of propofol to the
tubulin in presence of zinc chloride (Figures 12 and 13), there is complete
collapse of structure, the nature of the CD spectrum has completely changed
suggesting major changes in its overall conformation 26-30. For further
confirmation of results, we have also carried out time resolved fluorescence
study of tubulin in presence of anesthetic. Fluorescence lifetime is a very
sensitive parameter for analyzing the excited-state interactions and the local
environment present around the fluoropore. In this study, we have used DAPI as
fluoropore. DAPI (4′,6-Diamidino-2-phenylindole), a polycationic fluorescent
reagent, binding site in tubulin is located at the interface of both subunits
(alpha- and beta- tubulin). Its binding site is different from that of
colchicine, vinblastine, or taxol, does not interfere greatly with microtubule
polymerization.

Conclusion

Kinetics studies show
that propofol strongly affects polymerization of tubulin or self-organization
of microtubules. It is hypothesized that propofol’s effect is time- and dose-
dependent, and can be reversed when propofol is removed. Microtubule population
in presence of anesthetic propofol is not capable of carrying out collective
action. Microtubules do not self-organise by a reaction-diffusion process in
presence of propofol and do not communicate indirectly with each other.
Self-organizing patterns suggesting the potential for MTs to process
information do not form in the presence of anesthetic. Circular dichroism of
tubulin in presence of propofol suggests major changes in its overall
conformation. It is inferred that binding of anesthetics to tubulin protein
causes an alteration in secondary structure. TRFS study further supports the
change in secondary structure of tubulin protein when it binds with anesthetic
(propofol). We are not sure that assembly/polymerization of microtubule is the
best measure of microtubule activity relevant to consciousness. Also we have
not considered conditions ideal for quantum brain structures relevant to
consciousness. But our future endeavor would be to determine the conformation
of tubulin under conditions ideal for quantum brain structures relevant to
consciousness. In future we may discover quantum effects in microtubules
responsible for information processing..

Results and
Discussion                                                                        

Eon Spectrometer was
used at 350 nm for 1-2 hours 
Instrument                            
with the time interval of 1 minute at 37°C to monitor  the kinetics of microtubule assembly.
Polymerization of bovine brain tubulin was done by without usage and usage of
guanosine triphosphate (GTP). Graph show different effects in the presence and
absence of (GTP). In the presence of (GTP) the microtubule polymerization or
self organization was found to be stable to some extent and also less dynamic.
Microtubule was found to be less stable in the absence of  GTP.( GTP) also show other effects that are
not only enhance the of polymerization, but is also essentially required for
polymerization . Effects of taxol also seen on polymerization of tubule and
microtubule in the presence of GTP and glycerol are stabled in the presence of
taxol. The effect of propofol was seen on tubulin polymerization in the
presence of GTP and zinc ion  and
strongly affected with all three concentrations of propofol  with all three sets of experiments. It is
inferred that propofol (anesthetic) binds with hydrophobic pockets of tubulin
by weak force of  Vander waals London
dispersive force.Propofol effect is defined as dosage dependent and time . When
we remove propofol then it can be reversed. 
For the evaluation of the conformation and stability of tubulin protein
a technique is used that is called 
circular dichroism spectroscopy.Method is based on ?helix,?sheets and
random coil.By adding profol to tubulin in presence of Zinc chloride as result
a complete collapse of structure show complete change in overall
confirmation.DAPI not bind to tubulin heterodimer remains free so  it is prefer that binding of propofol with
tubulin rather  binding site than that of
DAPI and then DAPI transfer energy to neighbouring molecule.                                                                   
   

Conclusion                                                                                                                      

Experiments show that
polymerization of tubules or self organization of microtubles is strongly
effected by profol.By removing propofol the process can be reverse.In presence
of anesthetic propofol microtubule cannot perform collective action and do not
communicate with each other.From CD in the presence of propofol it show major
changes in overall confirmation.So, it is suggested that binding of anesthetics
to tubulin protein produce an alternation in secondary strcture.Change in
secondary strcture is due to binding with anesthetic.        

   Acknowledgement

The authors are
extremely grateful to Revered Prof. P. S. Satsangi, Chairman, Advisory Co
mMittee on Education, Dayalbagh Educational Institutions for incessant guidance
and encouragement. I am also grateful to Advance Instrumentation Research
Facility (AIRF), Jawaharlal Nehru University (JNU), New Delhi, for providing us
research facility.                                                            

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