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

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Control MEF Irradiated MEF Control HUH7 Irradiated HUH7

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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

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CTRL H2O2 IR+H2O2 IR

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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”