-
SCIENTIFIC REPORT
A NOVEL APPROACH TO REDUCE OXIDATIVE STRESS AT MOLECULAR AND
CELLULAR LEVEL WITH APPLICATIONS IN REGENERATIVE MEDICINE
PROJECT NO:139/2011
PHASE 1
15 .10 - 15.12. 2011
The effect of high density green photons(HDGP) irradiations on alkanes mixtures
We used in this study the term High Density Green Photons (HDGP) light (as in our previous
papers) due to the high density of photons provided by 1000 lumens of the LED which ensures a
strong impact of light with biological structures In this experimental set-up an alkanes mixture
(mineral oil) was used as substrate to be irradiated with HDGP. Under thermal degradation the alkanes
generate free radicals and accordingly are suited for the study of the oxido-reduction reactions, an
important class of cellular process.
Material and methods
To detect the possible modifications of alkanes mixtures a defined volume of mineral oil for each type
of measurement are placed in two dishes (for control and probe) and kept for 30 min into an electrical
oven at 165*C. The probe was exposed to HDGP.
After thermal treatment the mineral oil samples were analysed by liquid chromatography . . (Fig1)
The chromatographic results suggested that the HDGP irradiation induces an inhibition of free radicals
generation on thermal treated alkanes mixture and further a possible antioxidant effect.
-
Fig 1 Chromatograms of crude alkanes mixture (in blue), thermal degradated alkane mixture (in red)
and thermal degradated under green ligt (HDGP) irradiation (in green).
PHASE 2
16.12.2011 – 15.12.2012
The investigation of green light (HDGP) effects at cellular and molecular level .
1. Elaboration of experimental set-ups, materials and methods
a) Cell lines used in our experiments:
- HuH 7 Human hepatoma- immortalised cell line
- HEK Human Embryonic Kidney immortalised cell line
- MEF - Mouse Embryonic Fibroblast- immortalised cell line
- Human erythrocytes
- Human ADCS -Adipose Derived Stromal (stem) Cells
b) Isolation of ADSCs from human adipose tissue
- Adipose tissue ( obtained from abdomenoplasty) was washed with Hank’s balanced salt solution (HBSS , without phenol red , Gibco) containing penicillin/
streptomycin / amphotericin B (Sigma- Aldrich)
-
- Whashed adipose tissue was mechanically dispersed and washed 3-4x in HSSB and , HSBS eliminated by aspiration.
Medium from the final wash should be clear; if not, wash again.
- Dispersion of adipose tissue was achieved by collagenase digestion : with collagenase A type I (Gibco) in HBSS for 2 hrs on a shaker at 37* C.
and manually shaking the flasks vigorously for 5-10 sec every 15 min.
- On completion of digestion period ,the digested adipose tissue should have a “soup like “ consistency.
- Addition of FBS to stop collagenase activity.
- After digestion, the ability of lipid-filled adipocytes to float was used to separate them from stromal/stem fraction: collagenase digested tissue was dispensed
into 50 ml tubes and centrifuged at 4000xg for 10 min
- After centrifugation, the floating adipocytes, lipids and the digestion medium was aspirated
- The pellet was resuspended in red blood cells lysis buffer (Tris Base/ammonium chloride) for red cells elimination.
- After centrifugation,the cells were resuspended in HBSS containing fetal bovine serum (FBS- EuroClone- heat inactivated at 55*C for 30 min -)
- - For removing remaining undigested tissue and cell clumps the
suspension was passed through Falcon 100- -
(Becton Dickinson, San Jose, CA) .
- Cell suspension was applied to Histopaque -1077 gradient.
- After centrifugation cells at the gradient interface were collected and resuspended in DMEM/F12 ( Gibco) containing FBS , penicillin, streptomycin and
amphotericin B.
- Cells were added to 25 cm 2 culture flasks and cultured at 37 *C in CO2 atmosphere in humide air.
- After 7 days, attached cells were passaged by trypsinization and cultured in DMEM/ F12 containing FBS, penicillin and streptomycin.
The cells were characterized by light and fluorescence microscopy and flow
cytometry ( CD 90+, CD105+, CD14 - )
The microscopic and flow cytometry analysis of the cells isolated from human adipose
tissue confirmed that they are MSC/ADSCs (Fig 1 and 2)
-
Fig 1 Microscopic analysis of ADSCs in culture
-
Fig 2 Flow Cytometry characterization of isolated ADSCs
c) Cell cultures
All cell lines( excepting human blood erythrocytes and ADCS) were cultivated at 37*C,
5% CO2, in DMEM +10% FBS+ pen/strep/ amphotericin B
c a fost monitorizata prin 2 metode :
d) Cell proliferation was monitored by :
* Alamar Blue colorimetric method
* Cell counting with TALI Image Based Cytometer (Invitrogen)
2. Investigation of high-density green photons irradiation on water clusters in NaCl
solutions
The variety of biological effects induced by irradiations in cellular systems suggested the
investigation of water, as a common element of cellular structures. Within this context, we
developed a special geometrical experimental set-up to explore visible light, in particular green
light induced effects on water from a biological as well as physical viewpoint. We assume that
the green photon may react, via a novel type of field mediated interaction, with the water
aggregates found in normal cellular compartments, and modifies their cluster structures
Material and methods
-
The osmotic shock.
Solutions of 0,45 g% and 0,9 g%, irradiated with green light (λ=527 nm, intensity 3·105 Lx)
were used. As a receptor for measuring the irradiation effect we used red blood cell (RBC)
permeability in hypotonic media, the so-called osmotic shock.
After RBC lysis in hypotonic medium, the released hemoglobin was spectrophotometrically
determined at λ=550 nm. Samples which displayed at a preliminary checking an osmotic shock
value between 0.220-0.320 AU550 nm are used for the experiments with HDGP-irradiated NaCl
solutions. Physiological solutions (0.9%) of NaCl represent internal controls, 0.45% NaCl
hypotonic solutions Control (C) and HDGP-irradiated (I*) samples.
HDGP-irradiations are performed in air with a two Cree LED (100 Lumens) device, emitting
λ=527 nm, 3·105 Lx.Sets of 20 experiments are realized, with irradiations times of 15, 30, 45 and
60 min. Experiments were performed at room temperature.
Variations of temperature (
-
Irradiation times
(min.)
Controls, AU550 nm
± SD
HDGP-Irradiated*, AU550 nm
± SD
15 0.225 ± 0.040 0.181 ± 0.031
30 0.228 ± 0.021 0.170± 0.039
45 0.321 ± 0.052 0.246 ± 0.021
60 0.246 ± 0.035 0.158 ± 0.039
Table 1. Irradiation time dependence of the HDGP effect on hypotonic saline solutions
Chronoamperometry : We represent in table 2 the effect of GL-irradiation on ionic
mobility in a 0.9% NaCl solutions irradiated 60 min. by GL. As seen from the table, the current
density of the irradiated solution decreases from the value of 29.5 µA.cm-2 in the control to 17.74
µA.cm-2 in the irradiated sample with a corresponding decrease of ionic mobility, from 6.804·10-
10 (m.s-1/V.m-1) to 4.085·10-10 (m.s-1/V.m-1). This result correlates well with the osmotic shock
values, suggesting formation of large molecular aggregates of cluster type, which may decrease
ionic mobility.
Current Density
(µA·cm-2)
Ionic Mobility
(u++u-)·10-10 (m·s-1/V·m-1)
CONTROL
29.55 ± 5.11
6.804 ± 1.177
IRRADIATED
17.74 ± 5.10
4.085 ± 1.175
Table 2. Effect of 60 min GL-irradiation on current density & ionic mobility in a 0.9% NaCl solution
Impedance Spectroscopy : As seen from fig.1 the impedance value of the HDGP-
irradiated 0.9% NaCl solution is significantly lower than the control, thus correlating well with
-
the data recorded by chronoamperometry. The impedance values obtained with the blue and red
EM-visible bands present lower values, thus indicating the green band as the most efficient one.
Fig. 1: Impedance spectroscopy recording of a 0.9% NaCl solution, irradiated 45 min. in the green, blue
and red visible light band and of the nonirradiated control.
The experiments performed by chronoamperometry and impedance spectroscopy on ionic
mobility all correlate well with the results obtained on the osmotic shock, suggesting water
clusters formation in the respective HDGP-irradiated water solutions. We assume that the
green photon may react with the water aggregates found in normal cellular compartments, and
modify their cluster structures.
As a final point: the novelty of our experimental set-up rests on the method to detect
physical phenomena, using biological receptors. Due to their complex topological structure, their
resolution power surpasses by far the most sophisticated physical instruments. This might be a
concept of biological spectroscopy suggested long ago by Comorosan .
The results of this study were included and published in the following article :
R. Mitrica, I. Popescu, L. Paslaru, D. Badila, S. Polosan, L. Cristache, E. Ionescu, C. Tataru, S.
Comorosan “”High-density green photons effects on NaCl solutions detected by red blood
cells membranes Digest Journal of Nanomaterials and Biostructures Vol. 7, No. 1, January -
March 2012, p. 227 – 235 Impact factor=1,12
-
PHASE 3
16.12012-15.12.2013
A) Investigation by physical methods of high density green photons (HDGP) irradiation effects on physical and chemical parameters of enzymes molecules . (I)
Material and methods
Optical manipulation of proteins. The study was performed on α-amylase protein
(Sigma- Aldrich, 10085, EC 3.2.1.1, yeast, from Aspergillus oryzae, mol wt 51.103D).
HDGPG irradiation was performed with light-emitting-diodes (16V, 20W, 1000
lumens, EverRed Tronics, E 20 WG 120 C) mounted on ventilated copper radiators. A
monochromatic green light with absorption peak centered at λ-520nm was obtained, with
intensities up to 140mW/cm2, spectral width 10 nm.
Probes of 2 ml protein solution, placed on Petri dishes (φ=30 mm), were irradiated for
30 min with green light, 15 mW/cm2 intensity, 105 mW incident on the target area 7
cm2, placed at 7 cm from the source. The light beam was collimated to cover the entire
surface to be irradiated.
Physical methods
Atomic Force Microscopy (AFM).
Images were obtained with NTegra Prima AFM- using the Hybrid D mode. Samples were coated
on special microscopic slides with high adherence for glass (Corning BioCoat Coverslips) and
evaporated at room temperature.
Circular dichroism (CD)
Measurements were performed on Jasco J815 spectrometer, with water-cooled thermostated
Peltier device 0.10C accuracy, 163-900 nm range, in Hellma Suprasly quartz cuvette, 0.1 cm
path. Recordings of 50 μl protein solution at 00C.
Fluorescence spectroscopy
Fluorescence spectra were recorded on Edingburgh Instruments F900, at 900 angles, on Hellma
Suprasyl quartz cuvette, 1.0 cm path. Recordings of 1.5 ml protein solution.
Results and discussion
Atomic Force Microscopy.
We investigated some mechanical parameters, adhesion and elasticity of the α-amylase protein
under λ=520 nm irradiation, with AFM (atomic force spectroscopy). The adhesion forces
revealed, in the control sample, a single, limited distribution, with one peak at 0.8 nN on the 0-
1.5 Z axis (nN) domain, (fig.1, A). The adhesion forces revealed in the irradiated sample, a larger
distribution with two peaks at 0.65-0.9 nN on the 0-1.5 Z axis (nN) domain (fig.1, B). This
-
behaviour corroborates with zeta potential data, suggesting that the surface charges of the new
generated structures may induce a better surface adhesion.
A B
Fig.1 AFM images of the adhesion parameter for the control (A) and the HDGP-irradiated
protein (B).
The elasticity measurements revealed the predicted results: a more compact, denser
structure would be less elastic. In our AFM- images the control samples revealed a recorded
elasticity between 100- 450 (a.u.) on the Z axis with the peaks at 180-380 (a.u.) as compared
with the recorded elasticity between 200- 300 (a.u.) on the Z axis with the peaks at 230-270
(a.u.) for the irradiated samples (fig. 2 A, B).
-
Fig.2. AFM images of the elasticity parameter for the control (A) and the HDGP-irradiated
protein (B).
Circular dichroism.
Circular dichroism (CD) is the elective method for the study of secondary structure and
ellipticity of protein macromolecules, under the cellular conditions in which they actually
operate (solutions). The main unit used in proteins studies is MRE (mean residue ellipticity), the
molar ellipticity of the molecules divided by the number of monomer units, computed as MRE =
ϴ/10 rl, where ϴ is the recorded CD (mdeg) r- the number of amino-acids residues (511 for the
studied protein) and l- the path length (cm) of the specter cell. From it the α-helix content may be
computed by referring the mean residue ellipticity for a helix of r-amides to the mean residue
ellipticity of an infinite helix, or alternatively by fitting the recorded CD- curves with specific
Provencer- Glockner method for α-helix.
It is known that CD is a specific physical method to reveal the folds characteristics of β-
sheets in macromolecules. It was reported that the decrease of the CD negative peaks above 200
nm (specific for different extracts) is connected with the decrease of the folded protein fraction
in the amylase molecules .
In this study we investigated the CD- spectrum of the α-amylase protein under HDGP
irradiation. In our experimental set-up a sharp decrease of the CD signal for the HDGP irradiated
sample is reported within 215-220 nm range (fig. 3).
Fig.3- Circular dichroism of α-amylase protein under HDGP-irradiation.
This result is well corroborated with the SELCON 3 fitting of the experimental curve (fig.4).
-
Fig.4- MRE representation of the control and the HDGP- irradiated protein with the SELCON 3
fitting.
The fitting revealed a decrease of the α-helix content from 0.280 in the control to 0.165 in
the optically manipulated sample (table 1).
Table 1- Conformational parameters of α-amylase, in the control and the HDGP-manipulated
probe.
Sample -Helix -Sheet Bounded Turn Random Coil
Control 0.280 0.079 0.165 0.249
HDGP 0.165 0.105 0.231 0.294
Reference [16] 0.234 0.199 0.118 0.261
The decrease of the α-helix content means a different bonding of polypeptide chains. In
the irradiated samples this generates loosing, breaking and rebounding in different
conformations. The new conformations may be linked to the concept of disordered regions with
flexible loops that induce a different α-helix dynamics generating a high content of polar amino-
acids .
Fluorescence spectroscopy
. Fluorescence spectroscopy is an effective physical method for the study of molecular energy
levels concerned with electronic vibrational states. In this study we monitored the changes in the
intrinsic fluorescence of α-amylase protein, under irradiation with HDGP, in order to determine
the possible conformational changes occurring upon irradiation. We recorded two main
results. In the first place a decrease in the 340 nm fluorescence of the HDGP- irradiated protein
(fig. 5).
-
Fig. 5- Fluorescence spectra of α-amylase protein, under HDGP-irradiation.
This effect may be assigned to a partial polarization of the C=O bond into C-O- as well as
to a rigidization of the molecular degrees of freedom.
The decrease of the fluorescence signal at 340 nm, may be corroborated with the decrease of the
CD-signal in the optically irradiated sample, due to the partially disrupting of the secondary
structure.
In the second place the recorded fluorescence spectrum revealed a shift of the
fluorescence peak towards higher energies (from 340 nm to 334 nm), suggesting electric
interactions between the localized peptide structures. It is known that the red shift indicates
the exposure of tryptophan to the aqueous environment, whereas the blue shift indicates the
destabilization of hydrophobic regions and the concealing of tryptophan residues.
The results of this study evidentiated modifications of some physical and chemical
parameters of enzymes molecules under HDGP irradiations
B) Investigation of HDGP irradiations on cellular systems (I): Study of cells migration .
Material and methods
Cell cultures
MEF cell line was cultivated in Dulbecco’s modified Eagle’ medium (DMEM)(Gibco, Life
Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1%
antibiotic. Cells were seeded at different densities in 3.5 cm Petri dishes or 24 well plates and
incubated at 37˚C under a humidified atmosphere with 5% CO2.
-
High density green photon source and irradiation
Cell culture under different conditions were irradiated with a source of HDGP at different time
points. As a source of HDGP, a pure monochromatic light emitting diodes (LED) was used (16V,
20W, 1000 lumens, EverRedTronics, E 20 WG 120 C) Diodes were mounted on ventilated
copper radiators. A monochromatic green light with absorption peak centered at λ-520nm was
obtained, with intensities up to 140mW/cm2, spectral width 10 nm. Time of cells irradiation
varied between 1-60 min , at different intervals of time (e.g 2x5 minutes /24hrs of incubation)
Cell migration assay
A scratch assay model was used to determine cell migration. Briefly ,cells were seeded at the
same concentration in 3,5 cm Petri dishes. When cells reached confluence scratches were made
in cells monolayer with a sterile pipette tip. Irradition of cells was performed: 4 times x 5min /
36hrs. At the beginning of experiment images were taken and at regular intervals, the closure of
the schrach by migrating cells was microscopically monitorized. After 36hrs of incubations we
stained the cells with Coomassie blue and images were taken.
Results
To study the migration capacities of MEFs (controls and exposed to HDGP), a scratch model
experiment was used : scratches („wound gaps”) in cells monolayer were made and „healing „
of the gaps by cell migration and growth toward the center was monitored.
Microscop images were taken after 36 hrs. Comparing the images we evaluated the capaciy to
close the gaps and observed that the exposure to green light significantly activated the
MEFs migration capacity. (Fig 6)
MEF T0 MEF control at 36h MEF irradiated with HDGP at 36 h
Figure 4: Scratch assay in MEF cell line. Pictures were taken at T0 and after 36h
incubation for both controls and for irradiated samples
-
PHASE 4
16.12 2013 – 15.12. 2014
A) Investigation of HDGP irradiations on cellular systems (II): 1. The effect of HDGP irradiation on cell proliferation.
Material and methods
Cell cultures:
MEF and HUH7 cell line were cultivated in Dulbecco’s modified Eagle’ medium
(DMEM)(Gibco, Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum
(FBS) and 1% antibiotic. Cells were seeded at different densities in 3.5 cm Petri dishes or 24
well plates and incubated at 37˚C under a humidified atmosphere with 5% CO2.
High density green photon (HDGP) source and irradiation
Cell culture under different conditions were irradiated with a source of HDGP at different time
points. As a source of HDGP, a pure monochromatic light emitting diodes (LED) was used
(16V, 20W, 1000 lumens, EverRedTronics, E 20 WG 120 C) Diodes were mounted on ventilated
copper radiators. A monochromatic green light with absorption peak centered at λ-520nm was
obtained, with intensities up to 140mW/cm2, spectral width 10 nm. Time of cells irradiation
varied between 1-60 min , at different intervals of time (e.g 2x5 minutes /24hrs of incubation)
Cell proliferation assay:
Control and HDGP irradiated cells were incubated for diferent period of time and cell proliferation was
measured by two methods: using Alamar blue (Sigma –Aldrich) staining (assay cells were treated with
Alamar Blue and after 1-3h incubation the shift of color was determined spectrophotometricaly) or
using the Tali® Image Cytometer (Life Technologies).
Results
A series of cell proliferation experiments was performed. In all experiments a stimulation of
cell proliferation was observed; the degree of this stimulation was mainly function of
irradiation protocol. Irradiation for longer period of time (40-60 min) might induce an inhibition
of proliferation for certain cell type (data not shown).
In figure 1 we present the simulative effect of HDGP irradiations(4x5 min/48 hrs incubation) on
the proliferation of two cell lines: MEF and HUH 7.
-
Fig.1 Cell Proliferation assay
2.The effect of HDGP irradiation on cells submitted to oxidative stress.
Oxidative stress induction:Cell cultures treatment with hydrogen peroxide
For oxidative stress induction,cells were treated with different concentrations of hydrogen
peroxide in the presence (simultaneously or consecutive) or absence of green light irradiation for
different duration; right after the treatment, the culture medium was changed and cells incubated
at 37˚C. Usually, cells were processed for subsequent experiments after 24,48 or 72 hours of
incubation .During this incubation time ,supplimentar irradiations were performed.
The cellular response to oxidative stress was monitored by quantitative PCR (qPCR) for SOD
(superoxide dismutase)expression and by measuring the catalase activity
RNA isolation
0
100
200
300
400
500
600
700
800
900
Control MEF Irradiated MEF Control HUH7 Irradiated HUH7
REL
ATI
VE
CEL
L N
UM
BER
-
Total RNA was prepared from cell lines using Trireagent (Sigma, St. Louis, MO) according to
manufacturer’s instructions. The quantity and quality of the total RNA were assessed by
spectrophotometry with Nano Drop 1000 (Thermo Scientific, Arlington, TX). Samples with a
ratio 260/280 of 1.8-2.1 were used in downstream analysis.
cDNA synthesis and Quantitative PCR (qPCR):
First cDNA was obtained from 2 μg of total RNA using High Capacity cDNA Archive Kit (ABI,
Foster City, CA) in a total volume of 20μl. The final dilution of the samples was 2 ng/μl. Two-
step relative quantification was performed on 7300 Real time PCR (ABI, Foster City, CA) using
hydrolysis probes. Then qPCR amplification was carried out in triplicate for each sample in a
total volume of 25 μl at the following conditions: 95°C for 10 min, 95°C for 15 sec and 1 min at
60°C for 40 cycles. The level of each mRNA was normalized to reference gene hu18S (20x). We
determined fold changes within tumoral tissues compared with paired non-tumoral tissue. Data
were analyzed with SDS 1.4 software using comparative Ct method [2^(-delta delta Ct)]. The
tested genes were super oxide dismutase (SOD1) and catalase (CAT). As an endogenous control
we used 18S gene.
Catalase activity:
The catalase activity was assessed by Catalase Activity assay kit(BioVision, CA) . Samples and positive
control were prepared in the same manner. Briefly cells were homogenized with Assay Buffer,
centrifuged at 10000g for 15 min at 4°C. Supernatant was collected for assay. Standard curve was draw
by using different concentrations of H2O2: 0, 2, 4, 6, 8 and 10 nmols
Results
SOD expression .The results of the qPCR experiments are presented as an average of three
experiments conducted in the same conditions. For the analysis we used as calibrator the control
sample. Hydrogen peroxide addition and/ or HDGP irradiation modify cellular SOD gene
expression (Fig 2): in the hydrogen peroxide treated cells the fold change of SOD determined by
qPCR decreased compared with control sample (fold change 0.7, p-value=0.009). The irradiated
sample (IR) shows a greater increase in the fold change compared with control sample (fold
change 1.3, p-value=0.03) and also an increase of fold change compared to hydrogen peroxide
-
treated sample. In the sample simultaneously treated with H2O2 and irradiated with HDGP the
SOD gene expression is higher compared with control sample (fold change 1.1, p-value=0.2), but
smaller compared with the irradiated sample and increased compared to hydrogen peroxide
sample.
Fig.3 Quantitative polymerase chain reaction measurements of gene expression of SOD in
samples with various treatments (H202, irradiated or irradiated in the presence of H2O2)
compared with control (no treatment) relative to 18S. The values are expressed as mean of 3
independent replicates.
Catalase activity Compared with control sample the catalase activity was higher in hydrogen
peroxide treated and in irradiated samples. In the sample simultaneously treated with H2O2 and
irradiated with HDGP,the catalase activity was the highest determined among the studied
samples. (Fig3)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
control c H2O2 IR IR H2O2
-
Fig.3 Catalase activity
Discussion
The mechanisms underlying the effects of visible light on cells are incompletely understood.The
present study demonstrate that high density green photons (HDGP) irradiation stimulates
cellular proliferation and migration . Mitochondria could be a possible site for initial light
effects : increase of ATP production and consequently, stimulation and modulation in levels of
growth factors, cytokines and others parameters . In turn, these effects lead to increased cell
proliferation and migration with further modulations of cellular processes..
Our results concerning SOD gene expression and catalase activity indicate that HDGP
irradiations also induce changes in intracellular antioxidant processes and consequently
suggest possible antioxidant effects.
As a conclusion we consider that all this described cellular effects indicate that HDGP
irradiation may have beneficial bio-medical applications, particularly in regenerative
medicine field .
The research results concerning proliferation, migration rate and oxidative stress response of
cellular systems irradiated with HDGP were included in a published article :
L Paslaru, A Nastase , L Stefan, R Florea, A Sorop, E Ionescu, I Popescu, ,S.Comorasan
“STIMULATORY AND POSSIBLE ANTIOXIDANT EFFECTS OF HIGH DENSITY
GREEN PHOTONS (HDGP) ON CELLULAR SYSTEMS”Journal of Medicine and Life,
vol7,issue 4,oct-dec 2014, pp 619-622
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
CTRL H2O2 IR+H2O2 IR
CA
TALA
SE A
CTI
VIT
Y (M
U/M
L)
-
B) Investigation by physical methods of high density green photons (HDGP) irradiation effects on physical and chemical parameters of protein molecules . (2)
In this study we advanced the hypothesis that green light induces electric dipoles in the
protein, which interact with each other, generating conformational modifications toward a more
compact design, with different physical properties. ). In proteins the induced dipoles may
stabilize the fluctuating dipoles, associated mainly with the resonating valence of the peptide
bonds.
We emphasize that these induced dipoles are generated by radiation fields, i.e. by time-
dependent fields, not by static ones. Consequently, the induced dipoles oscillate and radiate
electromagnetic fields, on account of the energy absorbed from the original, incident radiation
field. Through the electromagnetic field they generate induced dipoles that may interact with
each other with a force which can be estimated. The effect of this interaction force causes
conformational changes, toward a more compact metastable state. Let us point that this is a
hypothesis. As such it should stand the experimental tests. In this work we covered our
hypothesis with the experimental results, which in all our set-ups supported it. Alternative
models may be considered, nevertheless, within an entire new theoretical approach.
Material and methods
Optical manipulation of proteins. The study was performed on α-amylase protein (Sigma-
Aldrich, 10085, EC 3.2.1.1, yeast, from Aspergillus oryzae, molwt 51.103D). GL irradiation was
performed with light-emitting-diodes (16V, 20W, 1000 lumens, EverRed Tronics, E 20 WG 120
C) mounted on ventilated copper radiators. A monochromatic green light with absorption peak
centered at λ-520nm was obtained, with intensities up to 140mW/cm2, spectral width 10 nm. Probes of 2 ml protein solution, placed on Petri dishes (φ=30 mm), were irradiated for 30 min
with green light, 15 mW/cm2 intensity, 105 mW incident on the target area 7 cm2, placed at 7
cm from the source. The light beam was collimated to cover the entire surface to be irradiated.
The physical methods used in this study:
Raman Spectroscopy
FTIR (Fourier transforms infrared spectroscopy)
Zeta potential
Cyclic voltammetry
Electrical Impedance Spectroscopy (EIS).
Results and discussion
-
Raman spectroscopy gives access to the vibrational and rotational molecular modes, which lie
usually in the (far) infrared range. The Raman spectrum of the α-amylase protein is dominated
by the amide band occurring at ≈1647 cm-1. This band is associated with α-helical
conformations. In the protein samples irradiated with HDGP the 1647 cm-1 peak is reduced
significantly (fig. 4).
Fig. 4- Raman spectra of α-amylase protein, under HDGP-irradiation.
This indicates that the strength of the optically-active mode of the α-helices is reduced in
irradiated samples, which corresponds to a reduction of the folding degree, consistent with the
previously presented circular dichroism and fluorescence results.
FTIR (Fourier transforms infrared spectroscopy)
The infrared spectra of proteins provide significant information concerning molecular
conformations within 668 cm-1, 1000 cm-1 and 1600-1700 cm-1 domains. The vibrational peak
at 668 cm-1 is assigned to OCN bending. In the HDGP-irradiated probe this peak is slightly
increased suggesting the stabilization of induced dipoles by the external field, with
generation of a metastable configuration. The vibrational peak around 1030 cm-1 is assigned to
C-O. In the protein structure the C-O vibrates within the polypeptide dipole context . The partial
stabilization of the dipoles in the peptide bond would change dramatically this context and
induce the significant difference between the control and HDGP-irradiated probe in the spectral
bands (fig.5).
-
Fig. 5- Fourier transforms infrared spectroscopy of α-amylase protein.
The frequencies of the amide I group between 1600-1700 cm-1 are assigned to C=O
streaching vibrations of the peptide bonds. The bands are generally characterized by a
deconvolution computation which yields α-helix parameters consistent with the theoretical
calculation and the recorded spectra . For the C=O streching bands the computations on the
control sample centered at 1652 cm-1 revealed an analytic area of 0.05. The HDGP-irradiated
sample centered at 1654 cm-1 revealed an analytic area of 0.025, which represents a clear
reduction of the α-helix. This result is consistent with the data from the Raman spectroscopy.
Zeta potential. It is a key physical parameter to measure surface charge on proteins. In this study we performed zeta potential measurements in order to determine possible modifications on
the surface charge of the α-amylase protein, under irradiations with HDGP. The main
experimental result revealed an extended surface charge of the protein macromolecule for the
irradiated probe, from -80 mV to +60 mV, as compared with -60 mV to +50 mV (fig. 6) for the
control.
-
Fig.6. Zeta potential distribution of -amylase protein recorded between -200 and +200 mScm-1.
It is known that polarization of the large molecules is distributed mainly on the surface.
Accordingly, the increase of the zeta potential on the protein surface electric charge
represents a clear indication of the polarization effects. A higher charge distribution would
imply a higher conductivity, confirmed by the experimental results (table 1).
Table 1- Zeta potential parameters for the control and the irradiated protein.
Sample Zeta potential (mV) Zeta deviation
(mV)
Conductivity (mScm-1)
P -7.46 21.4 15.9
P* -7.80 26.9 18.2
This effect would generate also a greater attraction between molecules, inducing molecular
aggregation.
Cyclic voltammetry study is focused on the phenomena that occur at the electrode/solution
interface under the influence of d.c. polarization. Fig. 7 presents the effects of HDGP irradiation
on the α-amylase protein in PBS, evaluated by cyclic voltammetry curves performed on the gold
surface as working electrode. In these potentiodynamic electrochemical measurements, which
are generally used to study the electrochemical properties of an analytic (α-amylase protein) in
solution (PBS), the working electrode potential is ramped linearly versus time, from -1500 mV to
+1500 mv vs. Ag/AgCl and then the potential is scanned back to -1500 mV. The current at the
working electrode is plotted versus the applied voltage to give the cyclic voltammogram trace.
When the potential is sufficiently positive for a species in solution, that may be oxidized (i.e.
electrons going from the solution to the electrode) and produce an anodic current as a peak or a
waveform. Similarly, on the return scan, as the working electrode potential becomes more
negative than the reduction potential of a species, reduction may occur to cause a cathodic
current (i.e. electrons flowing away from the electrode). By IUPAC convention, anodic currents
are positive and cathodic currents negative.
http://en.wikipedia.org/wiki/Voltammetryhttp://en.wikipedia.org/wiki/Electrochemistryhttp://en.wikipedia.org/wiki/Analytehttp://en.wikipedia.org/wiki/Working_electrode
-
Fig. 7. Cyclic voltammograms of -amylase protein, under HDGP-irradiation.
The recorded voltammograms show two distinct domains: the cathodic one, from -200
mV up to -1500 mV, corresponding to hydrogen evolution and an anodic one from -200mV up to
1500 mV, corresponding to the oxidation processes that occur on the electrode surface.
A possible mechanism of the electrode reactions that occur on the gold surface on the
cathode and anode domains, after applying a reversible linear voltage ramp between -1500 mV
to +1500 mV is outlined in fig.8.
(A)
-
(B)
Fig.8. A suggested mechanism of electrochemical processes on gold electrode, on cathode and
on anode domains, occurring after HDGP- irradiation.
For the optically manipulated sample a lower cathodic current for hydrogen evolution
reaction was recorded on the gold surface, suggesting an inhibition of the electrode process after
HDGP irradiation. This significant behaviour may be associated with the polarization of proteins
under the action of an external electric field. The polarized proteins may induce an easier
adsorption on the gold electrode surface due to electrostatic forces between the cathodic
polarized electrode and the polarized proteins. The new proteins orientations in the electric field
would generate a reduction in the active electrode sites for hydrogen evolution reaction, thus
causing a reduced flow of electrons transferred through the interface gold/solution and thus the
current fall (fig. 8.A - cathode domain).
On the forward curve of anodic domain, two oxidation waves at about 100 mV and 1250 mV
associated with two oxidation processes that take place on the gold electrode surface may be
observed for the control sample. The presence of the oxidation peaks on the reverse curve
suggests that the proteins were involved in an oxidation process on the forward curve, with the
formation of unstable compounds that undergoes oxidation steps, at 0 V and -600 mV during the
return curve (fig. 8.B - anode domain).
For the irradiated samples no oxidation/reduction peaks were observed, suggesting a good
stability of the proteins and implicitly of the gold surface. This again strengthens the idea of
polarized proteins, adsorbed on the surface, protecting the gold electrode surface (fig. 8.A -
anode domain). Although the proteins are adsorbed on the surface, they do not react under the
influence of the electric field (no important electron flow transferred at interface was observed),
reinforcing the idea that they are in a stable P* conformational form.
Electrical Impedance Spectroscopy (EIS). Is a perturbative characterization of the dynamics
of an electrochemical process. Electrochemical impedance is measured by applying an a.c.
potential to the electrochemical cell and then measuring the current through the cell. The applied
-
potential is a small amplitude sinusoidal excitation at open circuit potential (OCP) and the
response to this potential is an a.c. current signal. The impedance as a measure of the ability of a
circuit to resist the flow of electrical current is represented as a complex number. A common
presentation method is the Bode Plot. The absolute values of the impedance (|Z|) and the phase-
shift () are plotted with log frequency.
The a.c. potentials studies (EIS) by Bode diagrams highlight the protein polarization under
HDGP- irradiation. A sharp decrease of the protein solution impedance from 40 k to 2.5 k
(increase in conductivity) was observed, after the sample irradiation, (fig. 9,A).
The a.c. potential perturbation of the gold electrode in an electrolyte solution of α-amylase
protein in PBS, under HDGP- irradiation, on the frequency range from 106 – 103 Hz, is presented
as Bode diagrams in figure 9,B.
(A) (B)
Fig. 9. Bode diagrams, impedance modulus vs. frequency (A) and phase and HDGP vs.
frequency (B) of -amylase protein, under HDGP- irradiation.
The recorded phase angle, for high frequencies was about -25 suggesting a pseudo-
resistive behaviour for control sample. For the irradiated sample the recorded phase angle was
about -60 indicating a shift towards pseudocapacitiv behaviour, due to the orientation of
polarized proteins on the electrode surface.
EIS emphasizes major changes in electrolyte solution (α-amylase protein in PBS) after
irradiation, resulted in a rise in conductivity due to protein polarization under HDGP- irradiation.
In our study this electrochemical technique represents a significant physical argument for
our suggestions of proteins polarization.
Conclusions
Our work belongs to the new highlighted domain of optical manipulation of matter . We
advanced a main hypothesis: under the action of an external electric field ( irradiations with
HDGP : λ=520 nm), physical and accordingly chemical properties of proteins molecules are
modified : the fluctuating dipoles from the peptide bonds, suggested by the canonic resonating
valence bond theory, may be stabilized as induced dipoles.We suggested that these stabilized
-
dipoles may interact with each other within the polypeptide chain and generate new metastable
conformational modifications of the protein macromolecules.
We tentatively termed these novel metastable conformations as polarized P*-proteins and we
detailed the physical characteristics of the reported P*-protein by circular dichroism,
fluorescence, Raman and FTIR- spectroscopy, zeta potential, cyclic voltammetry, electrical
impedance spectroscopy and atomic force microscopy.
The polarized P*-protein revealed a specific conformational structure completely different from
the classic ones.
Since the polarized proteins (P*) may acquire new energetic profiles through the newly revealed
polarization effects (change in charge distribution and orbital configuration), they will act
differently through interactions of the type P-P*, P*-P*, P1*-P-P2* and so on. This may suggest a
different approach for the very active field of protein-protein interactions, particularly relevant
for domains as immunology, regenerative medicine, epigenetics or drug design.
The research results of this study were included in a article accepted to publication in
European Physical Journal- B http://dx.doi.org/10.1140/epjb/e2014-50717-8 Sorin Comorosan, Irinel Popescu, Silviu Polosan, Cristian Parvu, Elena Ionescu, Liliana Paslaru,
Marian Apostol “Conformational changes and metastable states induced in proteins by
green light”