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Transcript of Absorbance and Fluorescence Spectroscopies of Green Fluorescent Protein.docx
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ABSORPTION AND FLUORESCENCE SPECTROSCOPIES OF GREEN
FLUORESCENT PROTEINAlgodon SML, Tamon LU and Tutor MV
Absorption and fluorescence spectroscopies are both useful in characterizing
protein samples. This experiment aimed to employ the Bradford assay coupledwith absorption spectroscopy to determine the concentration of recombinant
green fluorescent protein (GFP) extract while fluorescent spectroscopy was done
to determine the effect of extreme heat and pH to the fluorescence intensity.
Absorbance readings of GFP, along with Bovine Serum Albumin protein standard,
were measured at 595 nm. Using the BSA calibration curve, the mean
concentration of the extract was calculated to be 1.658 mg/mL. The molar
extinction coefficient was then determined to be 0.4604
. In fluorescence
spectroscopy, 0.02 M protein was optimal for monitoring fluorescence. The
intensity (RFU) was observed at temperatures from 35C to 95C (increments of 10
C), at pH 1.40 to 3.0 using HCl and at pH 11.0 to 12.30 using NaOH. Recovery of
fluorescence was tested by re-exposing the protein to room temperature after heattreatment and by neutralizing the acid and base added. Results show that the
melting temperature of the GFP extract was 55C. A decreasing trend of
fluorescence was observed with increasing degree of all stress factors. The least
fluorescence was observed at highly acidic conditions. For the recovery, the
transition from 35C to room temperature (~23C) gave the highest recovery
(71.48%) while that of 95C to room temperature gave the second lowest (1.042%).
The basic condition had 10.86% recovery while the acidic condition gave the
lowest, 0.01839%. Overall, fluorescence was most affected by the highly acidic
environment.
I. Introduction
Spectroscopy deals with the interaction of radiated energy with matter resulting to the redirection
of the radiation or transitions between the energy levels of the atoms or molecules. It came from the
words spectrum, which pertains to a range of related qualities, and skopein, which means to
look. Spectroscopic data, which is represented by a spectrum, is characterized by the response of the
matter to electromagnetic radiation as a function of wavelength or frequency. Spectroscopic techniques
often utilize the absorption, emission or scattering of electromagnetic radiation by matter to quantitatively
and qualitatively assess the characteristics of the matter. The matter can be atoms, molecules, ions, or
solids. Spectroscopy is also used to elucidate the components of a substance since the spectral lines
obtained from conducting a spectroscopic technique are characteristic of elements. In the field of
biochemistry, the device used for the analysis related to spectroscopy is a spectrophotometer consisting
of electromagnetic radiation source, monochromator, sample holder and detector (Dinh, 2003).Due to the wide scope of spectroscopy, spectroscopic techniques have been classified into
different categories based on the type of electromagnetic radiation used, type of interaction between the
material and electromagnetic radiation and the type of material used. Two of the most common
spectroscopic techniques are absorption and emission spectroscopies which are both useful in analyzing
samples (Dinh, 2003).
Absorption spectroscopy refers to the absorption of light through interactions with the sample.
There are several types of absorption spectrometric techniques, such as ultraviolet-visible, infrared,
atomic, and x-ray absorption spectrometry. These techniques vary mainly on the electromagnetic
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wavelength used. In absorption spectroscopy, the sample absorbs energy (photon) as a function of
wavelength or frequency of light, generating an absorption spectrum. Absorption spectroscopy is
particularly useful for detecting the presence of a substance in a sample and determining the amount of
that substance in the sample. Analysis of data obtained by absorption spectroscopy of a sample to
determine the concentration of a substance entails the application of Beers Law which states that the
transmission (or transmissivity), T, of the light through a sample varies logarithmically with the product of
absorbance coefficient, , for a certain substance and the distance travelled by the light or the path
length, l(Hollas, 2004). From this, the equation for Beers Law isderived and is given by
(1)On the other hand, the emission spectroscopy of a chemical substance pertains to the emission
of electromagnetic radiation of a specific wavelength by an atom due to its transition from a higher energy
level to a lower energy level. The energy emitted by an atom is approximately equal to the energy
difference between the two energy levels. The occurrence of a wide variety of transitions and energy
difference associated with them creates the electromagnetic spectrum characteristic of an atom. Thus,
emission spectroscopy can be used to determine the presence of atoms in a substance of unknown
composition and the atoms comprising a molecule can be elucidated. One particular type of emission
spectroscopy is fluorescence spectroscopy, which deals with using light to excite molecules in a sample,
causing these molecules to emit light, usually visible light, that can be detected (Lakowicz, 2006).In this experiment, absorbance and fluorescence spectroscopies were conducted on green
fluorescent protein (GFP). The green fluorescent protein (GFP) is a fluorescent protein that can be
isolated from the Pacific jellyfish Aequoria victoria and many other marine organisms. Its fluorophore
originates from the internal Ser-Tyr-Gly sequence that is post-translationally modified to 4-(p-
hydroxybenzylidene)-imidazolidin-5-one (See Figure 1). However, it should be noted that fluorescence is
not an intrinsic property of the Ser-Tyr-Gly tripeptide. The cyclisation of the peptide and the oxidation of
the tyrosine residue results to the formation of p-hydroxybenzylidene-imidazolidone structure which is
responsible for the fluorescent phenotype (Yang et al., 1996). This fluorophore can exist in two resonant
forms: (1) with a partial negative charge on the benzyl oxygen of the tyrosine and (2) with the charge on
the carbonyl oxygen of the imidazolidone ring. These oxygen atoms form interactions with the basic
residues His148 (with Tyr66) and Gln94 and Arg96 (with the imidazolidone) that stabilize and allow
further delocalization of the charge on the fluorophore (Yang et al., 1996).
Figure 1. Fluorescent structure of GFP. The formation of the 4-(p-hydroxybenzylidene)-imidazolidin-5-
one structure of the Ser-Tyr-Gly tripeptide allows the protein to exhibit fluorescence (Yang et al., 1996).
Aside from spectroscopy, Bradford reaction was used to verify the concentration of the samplethrough comparison with a standard. The change in color of Bradford reagent when it binds to proteins
allows the sample to absorb light at 595 nm. The degree of absorption of light is directly proportional to
the concentration of protein in the sample. Different factors, such as temperature, basic and acidic
environments, that can affect the fluorescence of the sample, were also tested (Bradford, 1976).
The experiment aimed to determine the concentration of the GFP sample through Bradford assay
which involves absorption spectroscopy. Fluorescence spectroscopy was then employed to determine the
effects of stress factors particularly heat and extreme pH to the structure, function and, consequently, the
fluorescence of the protein sample.
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II. Methodology
Preparation of protein samples for absorption spectroscopy
The 31st elution fraction containing the protein of interest and which has the brightest
fluorescence under a UV transilluminator was subjected to absorption spectroscopy. The protein solution
was diluted with 20 mM Tris-Cl (pH 8).
Quantification of protein concentration through a Bradford reaction
A total of 10 mL of 1X Bradford reagent was prepared. Then, 13 500 L aliquots of Bradford
reagent was placed in 1.5 mL microcentrifuge tubes. A protein standard sample set (bovine serum
albumin or BSA) was also prepared in concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mg/mL in ddH2O.
Undiluted, 1:10 dilution and 1:100 dilution in Tris-Cl pH 8.0 of the test protein sample was also prepared.
Afterwards, 10 L of protein sample was added to the 500 L aliquots of Bradford reagent. The
absorbance of each sample was measured at 595 nm. Consequently, a calibration curve was obtained
from the observed absorbance of the protein standards and was used to determine the concentration of
the test protein samples.
Fluorescence intensity testThe fluorescence from 525-570 nm with excitation at 460 nm of 1 mL sterile ddH2O placed in a
plastic cuvette was measured. Subsequent additions of sufficient amount of protein to the 1 mL sterile
ddH2O were conducted to produce a 1 M, 5M and 10 M concentration of protein solution, the
absorbance values of which was also obtained at the same wavelength of light.
Thermostability test
For the thermostability test, 4 mL of the protein solution of unknown concentration was prepared.
Afterwards, 1 mL of the protein solution was placed into two identical cuvettes; the rest was stored for
other experiments. The fluorescence of the two samples (test and control) at 525-570 nm with excitation
at 460 nm was obtained. The control sample was kept at room temperature while the other samples were
subjected to 2 minute incubations at 35C, 45C, 55C, 65C, 75C, and 85C. The fluorescence of the
two samples at 525-570 nm with excitation at 460 nm was again measured. The test sample was
incubated at room temperature for 2 minutes after each reading. The fluorescence of the two samples at
525-570 nm with excitation at 460 nm was then measured to test for signal recovery. The temperature at
which 50% of fluorescence was lost and the temperature at which fluorescence is lost completely was
determined.
Resistance to acidic conditions
To test the resistance of the protein to acidic conditions, 1 mL of the protein solution was placed
into another cuvette. The fluorescence of the two samples (test and control) at 525-570 nm with excitation
at 460 nm was then measured. Subsequent additions of 1M HCl to the test sample were conducted to
obtain the following HCl concentrations: 1 mM, 2 mM, 5 mM, 10 mM, 20 mM and 40 mM. The
fluorescence of the two samples (test and control) at 525-570 nm (excitation at 460 nm) was thenmeasured after each treatment. The buffer HCl concentrations at which 50% fluorescence was lost and at
which fluorescence was lost completely was also determined. Finally, the acidic buffer conditions were
neutralized with a series of addition of NaOH (1 mM, 2 mM, 5 mM, 10 mM, 20 mM, and 40 mM) and
recovery of the fluorescent signal was tested after each treatment.
Resistance to basic conditions
For the resistance to basic conditions test, 1 mL of the protein solution was placed into another
cuvette. The fluorescence of the two samples (test and control) at 525-570 nm (excitation at 460 nm) was
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obtained. The control sample was kept at pH 7.5 while sufficient 1M NaOH was added in succession to
the test sample to get the following NaOH concentrations: 1 mM, 2 mM, 5 mM, 10 mM, 20 mM and 40
mM. The fluorescence of the two samples (test and control) at 525-570 nm (excitation at 460 nm) was
then measured after each treatment. The buffer NaOH concentrations at which 50% fluorescence was
lost and at which fluorescence was lost completely was also determined. Finally, the basic buffer
conditions were neutralized with the subsequent additions of HCl (1 mM, 2 mM, 5 mM, 10 mM, 20 mM,
and 40 mM) and recovery of the fluorescent signal was tested after each treatment.
III. Results
Absorbance Spectroscopy
Table 1. Mean absorbance values at 595 nm of
Bovine Serum Albumin (BSA) protein standard
added with Bradford reagent
BSA
Concentration
mg/mL
Mean Absorbance (at 595nm)
ActualA Corrected
B
0 0.511 0
0.2 0.558 0.047
0.4 0.711 0.200
0.6 0.827 0.316
0.8 0.923 0.412
1.0 0.997 0.486
1.2 1.108 0.597A
Individual absorbance readings are given in Appendix Supplementary Table 1B
Corrected values were obtained by subtracting the actual mean absorbance (0.511) of 0 mg/mL BSA inBradford reagent to the actual values of the different concentrations of BSA.
Table 1 shows the mean absorbance of the different concentrations of BSA in Bradford Reagent
at 595 nm. Notably, the absorbance increased with increasing concentration. The absorbance values of
those solutions were used to construct a standard curve for the Bradford reaction (See Figure 2 below)
and the equation of the line is also given. The curve had a positive slope and a regression coefficient (R2)
close to 1.
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Figure 2. Standard Curve for Bradford Reaction with BSA as the protein standard
The same relationship, which is the direct proportionality of absorbance to concentration, was
observed for the mean absorbance values of the GFP positive control and test sample given in Table 2.
As the GFP concentration is decreased by diluting the sample, absorbance also decreased. Unlike most
of the values, there are absorbance values that are greater than one for both GFP positive and test
sample and these values are italicized in Table 2 below. For instance, the actual mean absorbance of the
undiluted GFP positive (1.982) is greater than 1 even after correction (1.471). Another observation is that
the actual value for the 1:10 dilution of the GFP positive which is 1.056 became less than 1 after
correction (0.545). The same is true for the undiluted GFP test sample in which the 1.445 absorbance
value became 0.934. A negative corrected absorbance was obtained for the 1:100 dilution of the GFP test
sample after correction (-0.046) with a corresponding negative value for protein concentration. The valuesare underlined below.
Table 2. GFP concentrations of positive and test samples calculated using their mean absorbance
and the BSA protein standard curve
GFP Positive Control Test Sample
Mean Absorbance
(at 595 nm)
Calculate
d
Concentr
ation
(1x)C
e
E
Mean Absorbance
(at 595 nm)
Calculate
d
Concentr
ation
(1x)C
e
E
ActualA
CorrectedB
ActualA
CorrectedB
Undiluted
1.982 1.471 2.887 1.445 0.934 1.843 0.5066
1:10 1.056 0.545 10.87 0.5347 0.572 0.061 1.473 0.4141
1:100 0.546 0.035 9.677 0.3617 0.465 -0.046 -.6.128
Mean 10.27D 0.4482 1.658
D 0.4604
AIndividual absorbance readings are given in Appendix A Supplementary Table 1
BCorrected values were obtained by subtracting the actual mean absorbance (0.511) of 0 mg/mL BSA in
Bradford reagent to the actual values of the different concentrations of BSA.
y = 0.5146x - 0.0148R = 0.9904
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Absorbanceat595nm
BSA Concentration (mg/mL)
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CThe corrected absorbance values of the GFP solutions were used to derive the concentration.
DThe extreme values (marked with strikethrough) were not included in the calculation.
EIn calculating the molar extinction coefficients, the actual concentrations of the diluted solutions were
used, not the derived value for the stock (or the 1x). Also, the extreme concentration values were not
used to solve for e.
The standard curve was used to calculate the protein concentrations of the GFP control and test
samples shown in Table 2 (See Appendix A for the calculation). The mean concentration for the GFP
positive control and test sample were calculated to be 10.27 mg/mL and 1.658 mg/mL, respectively.
Table 2 also shows the calculated values for the molar extinction coefficient (e). The mean values for the
control and test sample, 0.4482
and 0.4604
respectively, are close to each other.
Fluorescence Spectroscopy
A. Intensity Test
Figure 3. Fluorescence Intensity of GFP at different concentrations. The fluorescence intensity was
measured using a fluorometer at a wavelength range of 525 nm to 570 nm. Three readings at each
concentration were obtained and the mean value was used to construct the graph. (See Appendix B for
the actual values).
The behavior of the graph in Figure 3 shows that fluorescence intensity increases with protein
concentration. The sample with a protein concentration of 0.02 uM highlighted red in Figure 2 gave anintensity that is intermediate of the values given by other samples.
0
20000
40000
60000
80000
100000
120000
0 0.02 0.04 0.06 0.08 0.1 0.12
FluorescenceIntensity(RFU)
GFP Concentration (uM)
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B. Thermostability Test
Figure 4. Effect of varying temperature to the fluorescence of GFP sample (0.2 uM). The same
fluorometer was used and the wavelength range is 525 nm to 570 nm. Three readings at each
temperature were obtained and the mean value was used to construct the graph. (See Appendix B for the
actual values).
^ The initial intensity represented by the leftmost data points (orange and blue diamonds) were measured
at room temperature (RT, 23C).
*The control was not subjected at varying temperatures. It was constantly at room temperature; however,
its fluorescence was also measured each time the test sample was read so it has readings for recovery.
**After subjecting the test sample at higher temperatures, it was allowed to recover at RT for 2 minutes
and its fluorescence was again measured. For the control, the same process was done but it was not
subjected to a different temperature; it remained at RT.
In Figure 4, the fluorescence intensity of the test sample decreased as the incubation
temperature was increased. Abrupt decrease in fluorescence was observed from 35C to 45C but it
became relatively uniform downstream of the graph. When GFP was allowed to recover at room
temperature (23C) every after exposure to higher temperature, fluorescence was not reset to the initial
value; in fact, the fluorescence readings after recovery also showed a decreasing trend. However, in
general, the fluorescence after recovery was slightly stronger than that with heat treatment. An exception
would be at 35C.
Notably, approximately 50% of the initial fluorescence of the test sample was lost at 55C;however, complete loss of fluorescence was not observed. The lowest intensity value was 73.54 RFU.
Relative to the test sample, the control, which was kept at room temperature, gave fluorescence
values that did not change drastically. The values ranged from 8000 to 11000 RFU while the test sample
reached below 100 RFU. Consistently, the control which was subjected at a lower temperature than all
the heat treatments had stronger fluorescence than the test sample.
Initial intensity(RT)^
0
2000
4000
6000
8000
10000
12000
0 20 40 60 80 100
FluorescenceIntensity(RF
U)
Temperature (C)
Test Sample
Test Sample (Recovery)**
Control*
Control (Recovery)**
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Figure 5. Effect of varying HCl concentration to the fluorescence of GFP sample (0.2 uM). The
same fluorometer was used and the wavelength range is 525 nm to 570 nm.Three readings at each HCl
concentration were obtained and the mean value was used to construct the graph. (See Appendix B for
the actual values).
^After reaching the 40mM HCl concentration, NaOH was added to successively lower the acid
concentration again to 20, 10, 5, 2, 1 and 0 mM.
*GFP was suspended in sterile distilled water as well as the test samples.
Based on Figure 5, a 1mM HCl concentration (pH 3) already caused a sudden decrease in
fluorescence intensity. As acid concentration was further increased up to 40 mM (pH 1.40), intensitycontinued to decrease at smaller increments. In contrast, the control showed a curved plot that is
gradually decreasing. Negative intensity values of the test sample, -2.4067, -3.1467 and -3.3933 RFU,
were obtained for 10, 20 and 40 mM HCl, respectively. When NaOH was added, original fluorescence at
10 to 40 mM HCl were attained; but, the sample did not recover its initial intensity upon complete
neutralization of the added acid. Before addition of the acid, the sample gave a fluorescence intensity
value of 11078 RFU and upon complete neutralization, the sample gave a fluorescence intensity value of
2.037 RFU. Reduction of fluorescence to 50% should be determined in between 0 to 1 mM HCl; however
this cannot be observed due to drastic changes in the fluorescence intensity. Complete loss of
fluorescence was not observed at all.
-2000
0
2000
4000
6000
8000
10000
12000
0 10 20 30 40 50
FluorescenceIntensity
(RFU)
HCl concentration (mM)
HCl added
Recovery (neutralizedby NaOH)^
Control (pH 7)*
HCl pH
1 mM = 3.0
2 mM = 2.70
5 mM = 2.30
10 mM = 2.0
20 mM = 1.70
40 mM = 1.40
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Figure 6. Effect of varying NaOH concentration to the fluorescence of GFP sample (0.2 uM). The
same fluorometer was used and the wavelength range is 525 nm to 570 nm. Three readings at each
NaOH concentration were obtained and the mean value was used to construct the graph. (See Appendix
B for the actual values).
^After reaching the 40mM NaOH concentration, HCl was added to successively lower the acid
concentration again to 20, 10, 5, 2, 1 and 0 mM.
*GFP was suspended in sterile distilled water as well as the test samples.
Similar to the observation under acidic conditions, the increasing base concentration greatly
decreased the exhibited fluorescence intensity of the sample as shown in Figure 6. However, the basic
condition, analogous to the control, gradually decreased the fluorescence intensity while the acidic
condition, as mentioned, immediately decreased the samples fluorescence intensity at 1 mM HCl (pH 3).Yet again, neutralization of the sample did not completely recover its fluorescence intensity. The sample
initially exhibited fluorescence intensity value of 11003 RFU and then exhibited 1195 RFU fluorescence
intensity upon complete neutralization. Also, original fluorescence was not attained at the other
concentrations unlike in the case of the acidic environment. Fluorescence reduction to 50% was
observed at 2 mM NaOH (pH 11.30). Complete loss of fluorescence was again not observed.
Table 3 summarizes the behaviour of the samples fluorescence at different conditions. Among
the three parameters, the highest percent recovery was obtained in the thermostability test specifically
after reverting the temperature from 35C to room temperature (23 C) while only 1.042% was recovered
at RT after exposure to the highest temperature used, 95C. The lowest percent recovery was obtained
when 40 mM HCl was neutralized with the same concentration of NaOH. The fluorescence recovery from
basic conditions was higher than from acidic pH.
0
2000
4000
6000
8000
10000
12000
0 10 20 30 40 50
FluorescenceIntensity(RFU)
NaOH concentration (mM)
NaOH added
NaOH neutralized byHCl^
Control (pH 7)*
NaOH pH
1 mM = 11.0
2 mM = 11.30
5 mM = 11.70
10 mM = 12.0
20 mM = 12.30
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Table 3. Loss and Recovery of GFP (0.2 uM) fluorescence under different environments
ConditionFluorescence Intensity
Percent RecoveryA
50% lost Initial (RFU) Recovered (RFU)
Temperature at ~55C
71.48% for 35C
(highest)
1.042% for 95C
(lowest)
Acid Cannot be determined 11078 2.037 0.01839 %
Base at ~2 mM (pH 11.30) 11003 1195 10.86 %
AIn calculating the percent recovery for temperature, the fluorescence intensity reading at RT after
exposure to heat treatment was compared with the initial intensity obtained at RT which is 11170 RFU.
IV. Discussion
Spectroscopy is the study of spectra. A spectrum (pl. spectra) is defined in physics as a
distribution of a characteristic of a physical system or a phenomenon. In the field of physical
biochemistry, various types of spectroscopic techniques are being used to study biological molecules,such as absorption, emission, mass, and nuclear magnetic resonance spectroscopy (van Holde et al,
1998). In this experiment, the concentration of the protein sample was determined through absorption
spectroscopy and the effect of different stress factors, specifically extreme heat and pH, were determined
through fluorescence spectroscopy.
Absorption Spectroscopy
Absorption spectroscopy deals with the transfer of electrons from the ground to excited
state. This transfer, which allows the absorption of energy at a particular wavelength, occurs due to the
presence of different molecular orbitals (See Figure 7). Electrons transfer from the ground to excited
state when the molecules of a sample are exposed to a light bearing an energy that allows an electronic
transition to occur. When this happens, some of the energy is absorbed. Electronic transitions occur
from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO),
and of the six possible transitions shown in Figure 7, only the leftmost two transitions are achievable
within 200-800 nm wavelength range (Laqua, 1988).
Figure 7. Different molecular orbitals and the transitions electrons can undergo. The transfer of
electrons from HOMO to LUMO due to excitation at a particular wavelength causes the absorption of
energy. In the figure, only the two leftmost transitions can be achieved in excitations using lights with
wavelength from 200-800 nm.
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Absorption spectroscopy is done through an absorbance spectrophotometer, an instrument that
measures the amount of light transmitted through the sample. The sample to be analyzed is placed in a
sample holder, called a cuvette, and then positioned in the instrument. A monochromatic light is then
allowed to pass through the solution and the amount of light that passed through the sample
(transmittance) or absorbed (absorbance) is measured by a light meter. In this type of spectroscopy, the
absorbance data is the one that is obtained (Hollas, 2004). The wavelength at which the absorption
occurs, as well as the degree of absorption, is recorded by an optical spectrometer. The spectrum
generated from the data is presented as a graph of absorbance (A) versus wavelength and is called the
absorbance spectrum. The absorbance values that can be obtained are between the range of 0 to 2, with
the zero value implying that no absorption occurred and a value of 2 implies that 99% absorption
occurred (Laqua, 1988). Since the other compounds in the sample, such as the solvent itself, can
interfere with the data by absorbing at the same wavelength as the sample to be analyzed, the
absorbance of the test solution is compared to a blank solution. The blank solution contains everything
that can be found on the test solution except the sample to be analyzed and the absorbance of the blank
solution is set at zero to correct the errors from the possible interference. Using the absorbance spectrum
obtained, the optimal wavelength, which is the wavelength that is most absorbed by the sample, can then
be determined. It is at the optimal wavelength that subsequent absorption spectroscopy of samples is
measured (Hollas, 2004).In order to analyze data from an absorption spectroscopy, it is important that the relationships
between the components of the technique must first be established. The relationship between the degree
of absorption of light (UV or visible light) and the properties of the material in a sample solution through
which light is passed through can be derived from Beer-Lambert Law, also known as Beers Law. The
equation for the relationship between transmission, absorbance coefficient and the path length is given by
(2)
However, since the absorbance coefficient of the sample can be obtained from the product of
molar absorptivity or extinction coefficient and the concentration of the sample, Equation 2 can be
rewritten as
(3)
Since the absorption is related to transmissivity by the following equation,
( ) (4)a linear relationship of absorbance with respect to the concentration of the sample can then be observed
and this is given by the equation:
(5)Since the absorbance of the sample can be measured and the concentration of the standard
protein and path length is known, a calibration curve can be obtained and the value of absorbance
coefficient can be calculated. Consequently, the unknown concentration of the test protein can be
calculated by substituting the values of the path length of the sample container and the absorbance
reading of the sample in conjunction with the calculated absorbance coefficient.
Before conducting the Bradford reaction on the protein sample, the sample was first diluted with
20 mM Tris-Cl pH 8.0 since absorption spectroscopy only applies to dilute solutions as can be seen later
on the limitations of Beers Law. The Tris-Cl solution was set at pH 8.0 since the physiological pH of the
green fluorescent protein is at this level. To quantify the protein concentration through a Bradford
reaction, a protein standard set of bovine serum albumin (BSA) in concentrations of 0, 0.2, 0.4, 0.6, 0.8,
1.0, and 1.2 mg/mL in ddH2O was prepared in order for a calibration curve to be constructed which will be
the basis for calculating the concentration of the protein in the test protein sample. BSA was used
because of its ability to increase intensity signal in total protein assays, and its stability and lack of
interaction with components of biochemical reactions. Aside from that, BSA is readily available to
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researchers because of its low cost and can be easily obtained at a high concentration by purifying
bovine blood, a product of the cattle industry. Technically, if the protein concentrations are the same, the
same absorbance values should be obtained for proteins provided that the same dilution buffer and same
stock solution of Bradford reagent was used for the assay. However, due to differences in concentration
of the protein, different absorbance values are obtained, but an equation showing the definite relationship
between concentration of the protein and the corresponding absorbance value can be obtained since the
concentration of the BSA is known and in turn, this can be used to determine the unknown concentration
of the protein sample (Doumas, 1975). A test protein sample set consisting of undiluted, 1:10 dilution, and
1:100 dilution with the Tris-Cl pH 8.0 was also prepared since, as have been mentioned, absorption
spectroscopy only applies to dilute solutions. The positive control used was asFP504, another fluorescent
protein which has an excitation maxima at 471 nm and 494 nm and emission maximum at 504 nm
(Concepcion, 2008). The positive control was necessary to confirm that the instrument used in reading
the absorbance of the samples is indeed functional. Following preparation of the protein standard set and
test protein sample, a Bradford reaction was conducted wherein 10 L of the protein sample and 500 L
aliquots of the Bradford reagent were mixed. It is necessary that the Bradford reagent is always mixed
with the protein sample at a larger amount since it is not known how much Bradford reagent will be
needed to react with the protein sample due to the fact that its concentration is unknown. If the Bradford
reagent is small in amount that it cannot bind all of the proteins present in the sample, then a less intensechange in color from the Bradford reaction will be observed and the concentration of the protein that will
be calculated will be smaller. On the other hand, if there is an excess of Bradford reagent, then the
Bradford reagent will be able to bind all of the proteins present in the sample and since it is the interaction
between the Bradford reagent and the protein in the sample that is detected by the spectrophotometer,
the absorbance obtained will still be accurate. The mixture of the Bradford reagent and protein test
sample was placed in a plastic cuvette for absorbance value reading since binding of the protein-dye
complex has been observed only with quartz cuvettes and may be eliminated by using either glass or
plastic cuvettes (Bradford, 1976).
In the experiment, Bradford assay was performed to determine the concentration of GFP samples
specifically by treating them with Bradford reagent and measuring their absorbance using a
spectrophotometer. This protein determination method is based on the shift in the absorbance maximum
of Coomassie Brilliant Blue G-250 in the reagent. Under acidic conditions, this dye exists in doubly
protonated tan to brown cationic form with absorbance maximum at 470 nm. Upon introduction to protein
samples, the dye binds with basic amino acids particularly to arginine through a combination of
hydrophobic interactions and heteropolar bonding (electrostatic interactions) (Georgiou et al., 2008). Van
der Waals forces and hydrophobic interactions may also be observed between the dye and the aromatic
amino acid residues (Trp, Tyr and Phe) (Compton and Jones, 1985). This binding then converts the dye
into its stable unprotonated blue form which absorbs maximally at 595 nm (Bradford, 1976).
A study has shown that the molar extinction coefficient of a dye-protein complex is constant over
a 10-fold concentration range (Spector, 1978). With only two unknown quantities left, Beers law can be
used to calculate concentration in terms of absorbance, the value obtained from the spectrophotometer.
The protein sample with the greatest concentration will exhibit the greatest absorption at 595 nm. This is
because the Coomassie Brilliant Blue G-250 dye binds proportionally to the proteins. The Bradford assayis a reproducible, easy and fast method to use for protein quantification. The dye binding process is
completed at approximately 2 minutes and the color can be maintained for an hour; thus, critical timing for
assay is not required. It is not prone to significant interference by cations (e.g. sodium or potassium) and
carbohydrates such as sucrose. Interfering color is only caused by significant amounts of detergents that
may remain on the glassware as well as strong alkaline buffering reagents (Bradford, 1976). Furthermore,
the protein-dye complex has a high extinction coefficient which corresponds to high-intensity absorption
of light (Singh et al., 2001). Despite these advantages, the Bradford assay also has limitations to be
considered. It cannot be used to quantify all proteins types since there are events wherein the protein
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sample assays may deviate chemically from Beers Law due to the proteins associa tion, dissociation and
interaction with the Bradford reagent and, sometimes, the solvent of the sample solution. In addition,
proteins that do not have Tyrosine and Tryptophan will not react properly with the Bradford reagent, and
hence, will not achieve the optimal tan colored solution that is required for the assay since the Bradford
reagent reacts strongly with these amino acids. Another limitation of this assay is the 2-10 mg protein
concentration limit. The Bradford assay is not valid for protein concentrations below or above this limit
because the plot that can be obtained from these data do not exhibit a linear graph anymore. This
therefore means that the Beers law cannot be applied anymore and possible errors, such as
underestimation for samples below 2 mg and overestimation for samples above 10 mg, can occur. As for
experiments where sample recovery is important, the Bradford assay is not recommended to be used
because the Bradford reagent has the capacity to denature the proteins due to its acidic property. In
relation this, the acidity of the Bradford reagent can also cause the proteins to aggregate, which can lead
to the sample being less quantified due to the unavailability of the residues that are supposed to react
with the reagent (Ninfa and Ballou, 1998).
At all cases, the absorbance readings of the control are greater than those of the test sample.
Expectedly, the mean stock concentration of the positive control which is 10.27 mg/mL is higher than that
of the test sample, which is only 1.658 mg/mL. This can be expected since the concentrations of the two
were not equalized.When the stock concentrations were calculated from both diluted and undiluted GFP and
asFP504 solutions, extreme values were obtained. For the positive control, the undiluted solution yielded
a value (2.887 mg/mL) that is way lower than the stock concentrations derived from 1:10 and 1:100
dilutions which are 10.87 mg/mL and 9.677 mg/mL. It was not included in the calculation of the mean
concentration so as not to incur a large error. This anomalous value may be attributed to the relatively
high corrected absorbance of the undiluted solution (1.471) which is probably out of the range wherein
absorbance is linearly related to concentration. In fact, it was the only corrected absorbance greater than
1. The range depends on the Bradford reagent and equipment but often, it does not include readings
greater than 1. The corrected absorbance values for the diluted solutions of asFP504 were only 0.545
(1:10) and 0.035 (1:100). In another case, the GFP test sample diluted a hundred fold (1:100) yielded a
negative concentration value which is -6.128 because the corrected absorbance is negative (-0.046).
Theoretically, obtaining a negative absorbance value is not feasible because it will lead to a negative
concentration which is not possible. However, there are instances that the absorbance readings will be
negative, simply because of the errors in the blank sample or due to very low concentration of the analyte
in the solution that it is beyond the detection limit of the Bradford reagent. The latter most likely explains
the negative value because a 100-fold dilution of the GFP test sample with a mean concentration of 1.658
mg/mL would lower the concentration to approximately 0.02 mg/mL which is way lower than the minimum
value that can be detected by the reagent (2 mg) given that 1 mL of the solution is in the cuvette
(equivalent to 0.02 mg protein in cuvette).
Proteins absorb a particular wavelength differently and so they differ in the molar extinction or
absorption coefficient. This value is also affected by factors such as solvent, solution composition, and
temperature and so environmental parameters were maintained all throughout the experiment to keep
constant and make the relationship of absorbance and concentration linear (Skoog et al., 2004). TheasFP504 and GFP have similar absorption capabilities and this is evident in their comparable calculated
molar extinction coefficients, 0.4482
and 0.4604
, respectively. This similarity in the degree of
absorbing light as well as in the degree of binding of the reagent to both proteins makes asFP504 a valid
positive control. A commercially available GFP is not necessary. Percent deviation was not calculated
because the molar extinction coefficients are expected to be slightly different since they come from
different proteins.
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To avoid anomalous data such as negative values for absorbance and overestimated or
underestimated values for concentration, the protein solution subjected to Bradford assay should be
diluted appropriately depending on the detection limit of the equipment and Bradford reagent used.
Beers Law only describes the absorbance of dilute solutions. At highly concentrated solutions, the
absorptivity is not constant and independent of concentration. It is already dependent on a quantity called
refractive index which may considerably vary at high concentrations. At high concentrations, the plot of
absorbance against concentration fails to follow a straight line because it bends toward the concentration
axis (x-axis) (Willard et al., 1981).
Fluorescence Spectroscopy
Fluorescence is an excitation and emission process in that molecules absorb energy in the form
of electromagnetic radiation of a certain wavelength and frequency. This electromagnetic radiation then
allows an electron to go from its ground state to excited state. The emission of a different wavelength and
frequency of electromagnetic radiation as the electrons return to its ground state is then detected and
processed as the fluorescence intensity. The wavelength of the emitted electromagnetic radiation is
different because energy is lost during the transitions, and as can be deduced from Plancks equation and
the relationship between the frequency and wavelength, the wavelength increases as the energy
decreases. Various factors such as the protein structure and rigidity, solution pH, temperature andconcentration of the proteins can affect the degree of fluorescence exhibited by the protein (Lakowicz,
2006).
In the experiment, 1 mL of sterile ddH2O placed in a plastic cuvette served as the blank. A blank
was necessary to eliminate the contributions of other interfering substances present in the sample that
may also fluoresce. The absorbance was measured at 525-570 nm because the fluorescent protein
exhibits maximum emission at that wavelength range (Yang, et al., 1996).
The increasing trend of fluorescence intensity as the protein concentration is increased is
expected because more protein molecules contribute to the signal detected. Usually, a linear correlation
between fluorescent protein concentration and fluorescence intensity is observed; but it should be noted
that the relationship between fluorescence and protein concentration is not really linear. In some cases, a
linear relationship between fluorescence intensity and protein concentration is observed at a certain
range of concentration specified for the protein used. More often than not, fluorescence is reduced at
higher protein concentrations due to reabsorption of the fluorescence photon by neighboring molecules,
limiting the linearity of fluorescence intensity against protein concentration to only that of dilute protein
sample solutions. In addition, at higher protein concentrations, self-quenching, inner-filtering and other
artifacts may occur, further reducing the intensity of fluorescence detected (Brandt, 2010). With these, an
intermediate concentration value was chosen to further monitor the fluorescence of GFP at different
environmental parameters (heat and pH). In this case, 0.02 uM GFP, which gave a fluorescence of 11657
RFU, was used. The highest intensity was 95313 (0.1 uM GFP) RFU while the lowest is 4561 RFU (0.01
uM GFP).
A. Thermostability Test
As for the thermostability test, it was observed that the fluorescence intensity of GFP decreasedas the incubation temperature increased. Although the -can structure of GFP is stable enough for it to
maintain fluorescence in the presence of detergents such as SDS and the fluorophore is highly protected
within the central helix near the geometric center, prolonged exposure to high temperature can
completely denature it and quench its fluorescence (Yang et al., 1996). When heat is added to the
system, proteins undergo denaturation, accounting for the observations made for the test sample. The
kinetic energy of the molecules within the protein increases as heat increases, and the increase in kinetic
energy leads to higher vibrations of the molecules, disrupting hydrogen bonds and non-polar hydrophobic
interactions within the protein. Hydrogen bonds and hydrophobic interactions are responsible for holding
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the protein together and disruption of these bonds may lead to loss of the secondary structure of the
protein which may lead to the eventual loss of biological functions of the protein such as fluorescence
(Lewis, 1926).
Complete heat denaturation of GFP is attained by exposing the protein at temperatures as high
as 90C in denaturing agents such as guanidium chloride (Yang et al., 1996). In general, the temperature
range of the treatment was not high enough to completely denature the protein and so complete loss of
fluorescence did not happen; only a decrease in fluorescence was observed which is expected at
temperatures greater than 30C. Fluorescence was not lost completely at 95C, which is possibly due to
the absence of other denaturing agents. However, a 150-fold decrease to 73.54 RFU (from 11170 RFU)
is a significant decrease. After exposure to higher temperature, the protein was allowed to recover for 2
minutes at room temperature (~23C). Failure to attain the initial intensity value proved that heat
denaturation is indeed irreversible (Yang et al., 1996).
The control sample, on the other hand, only exhibited a gradual decrease in fluorescence.
Theoretically, this should not be observed since control was maintained at room temperature. Most likely,
the cause is fluorescence quenching, which refers to any process that decreases the fluorescence
intensity of the sample. A common quencher is the oxygen molecule which is ubiquitously present in the
environment. Upon contact with the quencher, the fluorophore reverts to its ground state without emitting
a photon. Fluorescence intensity readings were acquired a couple of times and chances of oxygen havingcontact with the fluorophore increased with time (Lakowicz, 2006). Quenching might have also
contributed to decrease in fluorescence of the test samples but only causing minor effects.
The melting temperature (Tm) was determined to be 55C. According to Sauer et al. (2011), GFP
has a melting temperature above 65C. Despite this, the value from this experiment cannot be dismissed
because a protein may have several melting temperatures depending on the experimental conditions and
the analytical technique used in determining it. In addition, the value is within the usual melting
temperature of proteins which is 40C to 80C. Proteins having Tm higher than the range are already
thermophiles (Rajni and Mattiasson, 2003).
The range of decrease of the controls fluorescence intensity (11000 RFU to 8000 RFU) is
narrower than that of the test sample (11,000 RFU to 100 RFU). This shows that heat can drastically alter
the fluorescence of the protein.
B. pH-stability Test
Aside from protein concentration and temperature, pH also affects fluorescence, particularly the
spectral properties of the protein. It can alter the interactions between residues involved in fluorescence,
in particular the salt bridges, by changing the ionization state of those amino acids. The protein maintains
fluorescence over a wide pH range, specifically from 6 to 10 for Campbell and Choy (2001) and 8 to 11
for Haupts et al. (1998). However, it decreases at a pH lower than 6. This explains the decreasing trend
of fluorescence when it was subjected to acidic conditions. The pH range used was 1.40 to 3.0 and
relative to the range wherein fluorescence can be stably observed, the least acidic pH (1 mM HCl, pH 3.0)
is already low enough to cause the abrupt decrease in fluorescence intensity observed. In fact, at this pH
more than 50% of fluorescence was lost. From 11007 RFU, fluorescence lowered to 208 RFU when the
HCl concentration became 1 mM. Additionally, it has been observed that GFP fluorescence is highlysensitive to proton concentration such that it decreases to zero below pH 4. Negative intensity values
were even obtained in the experiment particularly for 10, 20 and 40 mM HCl (pH 2, 1.70 and 1.40,
respectively) proving that fluorescence had become very lowbeyond the detection limit of the
instrument. At low pH, the chromophore is observed to decrease absorption at 488 nm excitation
wavelength which is near the value that was used in this experiment that is 460 nm. Decrease in
absorption would result to a decrease in intensity of the fluorescence emitted. The decrease in
absorption is due to the protonation of the hydroxyl groups of a member of the active site, Tyr66
resulting
to a non-fluorescent species. This eradicates fluorescence quenching as the reason.
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According to Haupts et al. (1998), fluorescence of the protein also decreases at high pH values.
The basic pH values used as treatment were all greater than pH 11 and expectedly the fluorescence
intensity was observed to decrease. However, it should be noted that the decrease in fluorescence
intensity of the protein with increasing basicity was far more gradual than the decrease in acidic
conditions. From 11002 RFU, the lowest intensity attained was 3030 RFU at the highest NaOH
concentration which is 40 mM or pH 12.60. Fifty percent fluorescence loss needed a higher concentration
of NaOH, specifically at 2 mM NaOH or at pH 11.30. Also, complete loss of fluorescence was not
observed. Most likely, this subtle effect of basic pH on fluorescence is because of the chromophore being
protonated in a slightly different mechanism than what happens in acidic conditions. According to Haupts
et al. (1998), the protonated species is a result of the pH-independent internal protonation process
meaning that within the fluorescent protein, a proton is fluctuating or transferring between the hydroxyl
groups of Tyr66
and an internal proton binding site. The proton is not coming from the bulk solution. At low
pH, this internal protonation may occur at the same time, another proton from the bulk solution can again
cause protonation owing to the drastic decrease in fluorescence observed and the higher percent
recovery at basic condtions (10.86%) than at acidic environment (0.01839%).
At both pH fractions, recovery of the initial fluorescence intensity was not observed. This is
because treatment at extreme pH values causes mostly irreversible protonation of the active site and
alterations in the secondary structure of the protein.Other properties of the buffer that may be taken into account are salt concentrations, presence of
detergents and bovine serum albumin (BSA). High salt concentrations may induce aggregation of protein
samples. On the other hand, presence of detergents and BSA can interfere with fluorescence signal
intensity and other interactions within the protein. If some other substance is needed to be present in the
buffer, it is important that this substance is measured to take into account any background fluorescence
contributed by the added substance (Ugwu & Apte, 2004).
The highest percent recovery (71.48%) was obtained during the thermostability test; when the
protein was re-exposed to RT after heat treatment at 37C. Additionally, percent recovery due to removal
of 45C-, 55 C-, 65C- and 75C-heat treatments were all higher than the value due to removal of acid
and base (See Appendix Supplementary Table 7 for the actual values). This further proves the
robustness of the protein at temperatures lower than 80C. The recovery after exposure to 85C was
11.42% which is comparable with the basic environment, 10.86%. Recovery due to temperature reset
from 95C to room temperature and removal of 40 mM HCl yielded the low percent recovery values,
1.041 % and 0.01839% respectively. This indicates that exposure of protein to high temperature and
extremely acidic pH (~1.40) causes nearly irreversible denaturation or loss in fluorescence.
The degree of fluorescence recovery may vary among stress factors but in all cases the
percentage never reached 100%. Given that the fluorescence is primarily dependent on the primary
sequence of the protein rather than to its three-dimensional structure, partial recovery means that the
stress factors were able to alter the primary sequence of most of the protein molecules particularly in the
active sites. The alterations in the amino acid sequence hinder the protein from reverting to its native
conformation which means that the denaturation is irreversible. This claim is based on Anfinsens
theorem which states that in a given environment, the proteins native conformation is determined by the
primary structure, its amino acid sequence, at least for small globular proteins (Anfinsen, 1973).Irreversible heat denaturation occurs often at extreme values when protein molecules become
aggregated. A heat treatment that only causes unfolding of the native protein by weakening interactions
is reversible but once the protein molecules become fully denatured and their hydrophobic groups are
exposed, they tend to aggregate to hide those groups from the polar environment. To revert to the native
state, the aggregated protein should overcome an energy barrier. In the case of extreme pH, degradation
of acid-labile residues can cause cleavage of peptide bonds as well as alkaline hydrolysis at highly basic
environment.
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Quenching was observed in the control which meant that it could have contributed also to the
decrease in fluorescence of the test samples. To eliminate this error and to make sure that the stress
factor alone contributes to the recorded fluorescence intensity, exposure to common quenchers such as
oxygen may be avoided by covering the sample before and after taking the readings. Additionally, since
the extract was determined to be not pure, other quenchers might be present in the solution. Using a pure
sample will make the data more reliable.
V. Conclusion
The concentration of the GFP extract was successfully determined to be 1.658 mg/mL through
the Bradford assay. Results of fluorescence spectroscopy showed that an increase in temperature
starting from 35 C to 95C decreases the fluorescence intensity of the protein. The same effect was
observed as the environment became more highly acidic and more highly basic in particular at values
outside the stable range of the protein which is around pH 8 to 11. Removal of the stress factors resulted
to minimal recovery of fluorescence suggesting that extreme heat and pH conditions cause nearly
irreversible denaturation with the acidic environment having the most profound effect.
VI. References
Anfinsen, C. B.. "Principles That Govern The Folding Of Protein Chains." Science181.4096 (1973): 223-
230. Print.
Bradford, Marion M. "A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of
Protein Utilizing the Principle of Protein-Dye Binding."Analyical Biochemistry72 (1976): 248-254.
Web. 13 Oct. 2013.
Brandt, Mark. "Fluorescence Spectroscopy." Rose-Hulman Institute of Technology. N.p., 2010. Web. 9
Sept. 2013.
Campbell, Tessa N., and Choy, Francis Y.M. "The Effect of pH on Green Fluorescent Protein: a Brief
Review." Molecular Biology Today 2.1 (2001): 1-4. Web. 13 Oct. 2013.
http://www.horizonpress.com/backlist/mbt/v/v2/01.pdf.
Compton, S. J., and Jones, C. G. "Mechanism of dye response and interference in the Bradford protein
assay." Analytical Biochemistry151.2 (1985): 369-74. US National Library of Medicine National
Institutes of Health. Web. 13 Oct. 2013.http://www.ncbi.nlm.nih.gov/pubmed/4096375
Concepcion, Carla. "Cloning of the asFP504 gene in a mammalian expression vector and heterologous
expression of the monemerric cyan fluorescent protein (mCFP) variant in HEK293 and 4T1 cell
lines." Unpublished thesis (2008).
Dinh, Tuan. Handbook of spectroscopy. Weinheim: Wiley-VCH, 2003. Print.
Doumas, B T. "Standards for total serum protein assays--a collaborative study." Clinical Chemistry21.8
(1975): 1159-66. Print.
Georgiou, C.D., Grintzalis, K., Zervoudakis, G. and Papapostolou, I. "Mechanism of Coomassie brilliant
blue G-250 binding to proteins: a hydrophobic assay for nanogram quantities of proteins."
http://hoffman.cm.utexas.edu/courses/bradford_assay.pdfhttp://www.rose-hulman.edu/~brandt/Fluorescence/Fluorescence_Introduction.pdfhttp://www.horizonpress.com/backlist/mbt/v/v2/01.pdf.http://www.ncbi.nlm.nih.gov/pubmed/4096375http://www.ncbi.nlm.nih.gov/pubmed/4096375http://www.horizonpress.com/backlist/mbt/v/v2/01.pdf.http://www.rose-hulman.edu/~brandt/Fluorescence/Fluorescence_Introduction.pdfhttp://hoffman.cm.utexas.edu/courses/bradford_assay.pdf -
8/14/2019 Absorbance and Fluorescence Spectroscopies of Green Fluorescent Protein.docx
18/24
Analytical and Bioanalytical Chemistry 391.1 (2008): 391-403. US National Library of Medicine
National Institutes of Health. Web. 13 Oct. 2013.http://www.ncbi.nlm.nih.gov/pubmed/18327568
Haupts, Ulrich, Maiti, Sudipta, Schwille, Petra and Webb, Watt W.. "Dynamics of fluorescence fluctuations
in green fluorescent protein observed by fluorescence correlation spectroscopy." Proceedings of
the National Academy of Sciences of the United States of America 95.23 (1998): 13573-8. Print.
Hollas, J. Michael. Modern spectroscopy. 4th ed. Chichester: Wiley, 2004. Print.
Kaul, Rajni, and Mattiasson, Bo. Isolation and purification of proteins. New York: Marcel Dekker, 2003.
Print.
Lakowicz, Joseph R. Principles of fluorescence spectroscopy. 3rd ed. New York: Springer, 2006. Web. 13
Oct 2013. < http://link.springer.com/chapter/10.1007%2F978-0-387-46312-4_8#page-2>
Laqua, K. "Molecular Absorption Spectroscopy, Ultraviolet and Visible (UV/Vis)." Pure and Applied
Chemistry 60.9 (1088): n. pag. Internation Union of Pure and Applied Chemistry - IUPAC. Web.
14 Oct. 2013.
Lewis, P. S. The Kinetics of Protein Denaturation: Part III.The Influence of Neutral Salts on the Velocity
of the Heat Denaturation of Oxyhaemoglobin. Biochem J. 1926; 20(5):984992. Print.
Ninfa, Alexander J., and Ballou, David P. Fundamental laboratory approaches for biochemistry and
biotechnology. Bethesda, Md.: Fitzgerald Science Press, 1998. Print.
Sauer, Markus, Hofkens, Johan and Enderlein, J. Handbook of fluorescence spectroscopy and imaging
from single molecules to ensembles. Weinheim: Wiley-VCH, 2011. Print.
Spector, T. "Refinement of the Coomassie blue method of protein quantitation. A simple and linear
spectrophotometric assay for less than or equal to 0.5 to 50 micrograms of protein." Analytical
Biochemistry86 (1978): 142-146. Print.
Singh, K., Sandhu, G. K., Lark, B. S. and Sud, S. P. "Molar extinction coefficients of some carbohydrates
in aqueous solutions." Journal of Physics 58.3 (2002): 521-528. Indian Academy of Sciences.
Web. 13 Oct. 2014.
Skoog, D.A., West, D.M., James Holler, F. and Crouch, S.R. Fundamentals of Analytical Chemistry. 8th
edition. Brooks/Cole. 2004. p. 716, 723, 729.
Ugwu, Sydney O., and Apte, Shireesh P. The Effect of Buffers on Protein Conformational Stability. USA:
Pharmaceutical Technology, 2004. Print.
Weijers, Mireille, Barneveld, Peter A., Cohen Stuart, Martien A., and Visschers, Ronald W. "Heat-induced
denaturation and aggregation of ovalbumin at neutral pH described by irreversible first-order
kinetics." Protein Science 12 (2003): 2693-2703. US National Library of Medicine National
Institutes of Health. Web. 13 Oct. 2013.
http://www.ncbi.nlm.nih.gov/pubmed/18327568http://www.ias.ac.in/pramana/march2002/P5435.PDFhttp://www.ias.ac.in/pramana/march2002/P5435.PDFhttp://www.ncbi.nlm.nih.gov/pubmed/18327568 -
8/14/2019 Absorbance and Fluorescence Spectroscopies of Green Fluorescent Protein.docx
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Willard, H.H., Merritt, L.L. Jr., Dean, J.A. and Settle, F.A. Instrumental Methods of Analysis. 6th edition.
Litton Educational Publishing, Inc. 1981. p.19, 66-69, 72-73, 75.
Yang, Fan, Moss, Larry G. and Phillips, George N. "The Molecular Structure Of Green Fluorescent
Protein." Nature Biotechnology14.10 (1996): 1246-1251. Print.
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VII. Appendix
A. Absorption Spectroscopy
1. Raw Data
Supplementary Table 1. Absorbance readings of BSA standard and GFP positive
control and test samples all added with Bradford reagent
Absorbance
BSA concentration
mg/mLReading 1 Reading 2 Reading 3 Mean
0 0.51 0.511 0.512 0.511
0.2 0.558 0.558 0.559 0.558333
0.4 0.709 0.711 0.712 0.710667
0.6 0.826 0.827 0.827 0.826667
0.8 0.922 0.923 0.924 0.923
1 0.997 0.997 0.997 0.997
1.2 1.105 1.109 1.111 1.108333
GFP Positive control
Undiluted 1.982 1.982 1.982 1.982
10^-1 1.055 1.056 1.056 1.055667
10^-2 0.54 0.547 0.551 0.546
GFP Test sample
Undiluted 1.443 1.444 1.447 1.444667
10^-1 0.572 0.572 0.572 0.572
10^-2 0.463 0.465 0.466 0.464667
2. Sample Calculations
Determination of Protein Concentration of GFP samples
wherein c is concentration (1x) in mg/mL, a is the corrected absorbance at 595 nm and df is the dilution
factor.
GFP test sample (1:10 dilution)
Determination of mean molar extinction coefficient (e) for GFP
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wherein eis the molar extinction coefficient in mL*mg-1
cm-1
, c is the concentration (1x) in mg/mL, a is
the corrected absorbance at 595 nm and lis the path length which is in this case 1 cm.
Positive control (asFP504)
GFP Test Sample
B. Fluorescence Spectroscopy
1. Raw Data
Intensity Test
Supplementary Table 2. Fluorescence intensity readings at varying concentrations of the protein
Protein Concentration (uM) Reading 1 Reading 2 Reading 3 Mean CorrectedA
0 144.54 144.57 144.89 144.6666667 0
0.01 4562.87 4561.92 4558 4560.93 4416.263
0.05 37336.83 37294.26 37289.56 37306.88333 11656.69
0.1 95383.35 95475.84 95514.23 95457.80667 37162.22
0.02 11844.37 11793.78 11765.91 11801.35333 95313.14A
Corrected values were obtained by subtracting the mean absorbance (144.67) of 0 mg/mL protein to the
mean values of the different concentrations.
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Thermostability Test
Supplementary Table 3. Fluorescence intensity readings of control at varying temperatures
Temperature
(OC)
A
Control
Reading
1
Reading
2
Reading
3Mean
Corrected
Initial 10990.12 10996.55 11005.14 10997.27 10852.6
35 10693.29 10675.37 10647.45 10672.03667 10527.37
RT 10365.38 10341.64 10313.8 10340.27333 10195.61
45 10060.99 10050.88 10045.03 10052.3 9907.633
RT 9938.79 9951.25 9959.11 9949.716667 9805.05
55 9675.74 9682.59 9683.28 9680.536667 9535.87
RT 9618.36 9613.82 9614.19 9615.456667 9470.79
65 9454.24 9452.51 9442.99 9449.913333 9305.247
RT 9203.29 9194.41 9184.43 9194.043333 9049.377
75 9198.63 9197.4 9200.08 9198.703333 9054.037
RT 9157.66 9149.64 9149.95 9152.416667 9007.75
85 8886.46 8876.53 8869.87 8877.62 8732.953
RT 8611.8 8614.87 8616.5 8614.39 8469.723
95 8661.83 8654.62 8633.15 8649.866667 8505.2
RT 8418.48 8410.7 8405.22 8411.466667 8266.8A
RT stands for room temperature and is approximately 23C.BCorrected values were obtained by subtracting the mean absorbance (144.67) of 0 mg/mL protein to the
actual fluorescence intensity means.
Supplementary Table 4. Fluorescence intensity readings of test sample at varying temperatures
Temperature
(OC)
Test
Reading
1
Reading
2
Reading
3Means
Corrected
Initial 11343.26 11346.35 11253.34 11314.31667 11169.6535 9135.38 9120.95 9096.9 9117.743333 8973.077
RT 8117.59 8129.65 8140.4 8129.213333 7984.547
45 6631.49 6686.6 6730.55 6682.88 6538.213
RT 6909.53 6908.49 6904.72 6907.58 6762.913
55 5129.47 5256.22 5318.98 5234.89 5090.223
RT 5635.38 5644.05 5661.25 5646.893333 5502.227
65 3849.42 3915.11 3999.11 3921.213333 3776.547
RT 4447.06 4476.73 4498.45 4474.08 4329.413
75 2513.55 2664.44 2740.2 2639.396667 2494.73
RT 3168.85 3186.91 3204.52 3186.76 3042.093
85 1008.81 1097.07 1143.26 1083.046667 938.38RT 1415.63 1422.15 1424.1 1420.626667 1275.96
95 213.56 223.71 217.35 218.2066667 73.54
RT 259.25 261.54 262.31 261.0333333 116.3667A
Corrected values were obtained by subtracting the mean absorbance (144.67) of 0 mg/mL protein to the
actual fluorescence intensity means.
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Resistance to Acidic Conditions
Supplementary Table 5. Fluorescence intensity readings of test sample at varying acidic pH
HCl
Concentration
(mM)
Reading
1
Reading
2
Reading
3Mean
CorrectedB
0 11241.26 11224.04 11201.6 11222.3 11077.63
1 373.07 349.82 336.82 353.2366667 208.57
2 225.62 224.47 223.76 224.6166667 79.95
5 148 146.93 146 146.9766667 2.31
10 142.33 142.25 142.2 142.26 -2.40667
20 141.53 141.49 141.54 141.52 -3.14667
40 141.28 141.28 141.26 141.2733333 -3.39333
40 141.28 141.28 141.26 141.2733333 -3.39333
20A
142.56 142.5 142.39 142.4833333 -2.18333
10
146.02 145.81 145.87 145.9 1.233333
5
146.6 146.26 146.11 146.3233333 1.656667
2
146.03 145.74 145.65 145.8066667 1.14
1A 145.82 145.62 145.55 145.6633333 0.996667
0
146.77 146.7 146.64 146.7033333 2.036667A
Sample is neutralized with NaOH to attain desired acid concentration.BCorrected values were obtained by subtracting the mean absorbance (144.67) of 0 mg/mL protein to the
actual fluorescence intensity means.
Resistance to Basic Conditions
Supplementary Table 6. Fluorescence intensity readings of test sample at varying basic pH
NaOH Concentration
(mM)Reading 1 Reading 2 Reading 3 Mean Corrected
B
0 11154.17 11149.5 11138.41 11147.36 11002.691 9612.89 9470.2 9093.94 9392.343333 9247.677
2 5561.27 5149.61 4888.01 5199.63 5054.963
5 4135.68 4116.89 4106.98 4119.85 3975.183
10 3963.4 3962.4 3961.19 3962.33 3817.663
20 3766.92 3768.76 3769.84 3768.506667 3623.84
40 3193.14 3173.74 3158.83 3175.236667 3030.57
40
3193.14 3173.74 3158.83 3175.236667 3030.57
20A
2707.65 2702.87 2697.97 2702.83 2558.163
10
2225.17 2138.25 2085.01 2149.476667 2004.81
5
1507.55 1505.67 1501.48 1504.9 1360.233
2
A
1389.4 1392.23 1394.7 1392.11 1247.4431
A 1386.96 1384.98 1382.04 1384.66 1239.993
0
1339.47 1339.49 1339.46 1339.473333 1194.807A
Sample is neutralized with HCl to attain desired base concentration.BCorrected values were obtained by subtracting the mean absorbance (144.67) of 0 mg/mL protein to the
actual fluorescence intensity means.
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2. Percent Recovery
hermostability test
Supplementary Table 7. Percent recovery after removal of heat treatment
Temperature
(C)
Recovered
RFU
%
RecoveryA
35 7984.547 71.4843
45 6762.913 60.54723
55 5502.227 49.26051
65 4329.413 38.76051
75 3042.093 27.23535
85 1275.96 11.42346
95 116.3667 1.041811A
The initial fluorescence intensity used to calculate is 11169.65 RFU.
Stability test in acidic conditions
Stability test in basic conditions