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Algodon 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 coupled with 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 heat treatment 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

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 is derived 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-(phydroxybenzylidene)-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-5one 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 sample through 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.

II. Methodology Preparation of protein samples for absorption spectroscopy st The 31 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 test The 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 then measured 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

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 0 0.2 0.4 0.6 0.8 1.0

Mean Absorbance (at 595nm) Actual 0.511 0.558 0.711 0.827 0.923 0.997

Corrected 0 0.047 0.200 0.316 0.412 0.486

1.2 1.108 0.597 Individual absorbance readings are given in Appendix Supplementary Table 1 B Corrected 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. 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) 2 and the equation of the line is also given. The curve had a positive slope and a regression coefficient (R ) close to 1.

0.7 0.6 Absorbance at 595 nm 0.5 0.4 0.3 0.2 0.1 0 0 -0.1 0.2 0.4 0.6 0.8 1 1.2 1.4 BSA Concentration (mg/mL) y = 0.5146x - 0.0148 R = 0.9904

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 values are 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 Mean Absorbance (at 595 nm) Actual

Test Sample e


Calculate d Concentr ation C (1x) 2.887 10.87

Mean Absorbance (at 595 nm) Actual



Calculate d Concentr ation C (1x) 1.843 1.473


Undil uted 1:10

1.982 1.056

1.471 0.545

1.445 0.5347 0.572

0.934 0.061

0.5066 0.4141

1:100 0.546 0.035 9.677 0.3617 0.465 -0.046 -.6.128 D D Mean 10.27 0.4482 1.658 0.4604 A Individual absorbance readings are given in Appendix A Supplementary Table 1 B Corrected 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.


The corrected absorbance values of the GFP solutions were used to derive the concentration. The extreme values (marked with strikethrough) were not included in the calculation. E In 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 Fluorescence Spectroscopy A. Intensity Test 120000 Fluorescence Intensity (RFU) 100000 80000 60000 40000 20000 0 0 0.02 0.04 0.06 0.08 GFP Concentration (uM) 0.1 0.12 and 0.4604 respectively, are close to each other.

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 an intensity that is intermediate of the values given by other samples.

B. Thermostability Test 12000 Initial intensity (RT)^

Fluorescence Intensity (RFU)


8000 Test Sample 6000 Test Sample (Recovery)** Control* 4000 Control (Recovery)**


0 0 20 40 60 Temperature (C) 80 100

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.

12000 HCl added 10000 Fluorescence Intensity (RFU) 8000 6000 4000 2000 0 0 -2000 10 20 30 40 50 Recovery (neutralized by NaOH)^ Control (pH 7)*

HCl 1 mM 2 mM 5 mM 10 mM 20 mM 40 mM

= = = = = =

pH 3.0 2.70 2.30 2.0 1.70 1.40

HCl concentration (mM)

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), intensity continued 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.

12000 Fluorescence Intensity (RFU) NaOH added 10000 8000 6000 4000 2000 0 0 10 20 30 NaOH concentration (mM) 40 50 NaOH neutralized by HCl^ Control (pH 7)*

NaOH 1 mM 2 mM 5 mM 10 mM 20 mM

= = = = =

pH 11.0 11.30 11.70 12.0 12.30

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.

Table 3. Loss and Recovery of GFP (0.2 uM) fluorescence under different environments Fluorescence Intensity A Condition Percent Recovery 50% lost Initial (RFU) Recovered (RFU) 71.48% for 35C (highest) Temperature at ~55C 1.042% for 95C (lowest) Acid Cannot be determined 11078 2.037 0.01839 % Base

at ~2 mM (pH 11.30)



10.86 %

In 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.

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

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 intense change 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 u nknown 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 assay is 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

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). The asFP504 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.

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 and concentration 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 decreased as 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

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 having contact 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 T m 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 highly sensitive 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 low beyond 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 66 absorption is due to the protonation of the hydroxyl groups of a member of the active site, Tyr resulting to a non-fluorescent species. This eradicates fluorescence quenching as the reason.

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 66 groups of Tyr 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.

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." Science 181.4096 (1973): 223230. 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 Biochemistry 72 (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. Compton, S. J., and Jones, C. G. "Mechanism of dye response and interference in the Bradford protein assay." Analytical Biochemistry 151.2 (1985): 369-74. US National Library of Medicine National Institutes of Health. Web. 13 Oct. 2013. 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 Chemistry 21.8 (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."

Analytical and Bioanalytical Chemistry 391.1 (2008): 391-403. US National Library of Medicine National Institutes of Health. Web. 13 Oct. 2013. 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. <> 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 Biochemistry 86 (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. <>

Willard, H.H., Merritt, L.L. Jr., Dean, J.A. and Settle, F.A. Instrumental Methods of Analysis. 6 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 Biotechnology 14.10 (1996): 1246-1251. Print.


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/mL 0 0.2 0.4 0.6 0.8 1 1.2 Undiluted 10^-1 10^-2 Undiluted 10^-1 10^-2 2. Sample Calculations Determination of Protein Concentration of GFP samples Reading 1 0.51 0.558 0.709 0.826 0.922 0.997 1.105 1.982 1.055 0.54 1.443 0.572 0.463 Reading 2 0.511 0.558 0.711 0.827 0.923 0.997 1.109 1.982 1.056 0.547 1.444 0.572 0.465 Reading 3 0.512 0.559 0.712 0.827 0.924 0.997 1.111 1.982 1.056 0.551 1.447 0.572 0.466 Mean 0.511 0.558333 0.710667 0.826667 0.923 0.997 1.108333 1.982 1.055667 0.546 1.444667 0.572 0.464667

GFP Positive control

GFP Test sample

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

wherein e is the molar extinction coefficient in mL*mg cm , c is the concentration (1x) in mg/mL, a is the corrected absorbance at 595 nm and l is 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 A Protein Concentration (uM) Reading 1 Reading 2 Reading 3 Mean Corrected 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 95313.14 A Corrected values were obtained by subtracting the mean absorbance (144.67) of 0 mg/mL protein to the mean values of the different concentrations.

Thermostability Test Supplementary Table 3. Fluorescence intensity readings of control at varying temperatures Control Temperature B Reading Reading Reading Corrected O A ( C) Mean 1 2 3 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 8266.8 A RT stands for room temperature and is approximately 23C. B Corrected 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 Test Temperature A Reading Reading Reading Corrected O ( C) Means 1 2 3 Initial 11343.26 11346.35 11253.34 11314.31667 11169.65 35 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 938.38 RT 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 116.3667 A Corrected values were obtained by subtracting the mean absorbance (144.67) of 0 mg/mL protein to the actual fluorescence intensity means.

Resistance to Acidic Conditions Supplementary Table 5. Fluorescence intensity readings of test sample at varying acidic pH B HCl Corrected Reading Reading Reading Concentration Mean 1 2 3 (mM) 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 A 40 141.28 141.28 141.26 141.2733333 -3.39333 A 20 142.56 142.5 142.39 142.4833333 -2.18333 A 10 146.02 145.81 145.87 145.9 1.233333 A 5 146.6 146.26 146.11 146.3233333 1.656667 A 2 146.03 145.74 145.65 145.8066667 1.14 A 1 145.82 145.62 145.55 145.6633333 0.996667 A 0 146.77 146.7 146.64 146.7033333 2.036667 A Sample is neutralized with NaOH to attain desired acid concentration. B Corrected 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 B Reading 1 Reading 2 Reading 3 Mean Corrected (mM) 0 11154.17 11149.5 11138.41 11147.36 11002.69 1 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 A 40 3193.14 3173.74 3158.83 3175.236667 3030.57 A 20 2707.65 2702.87 2697.97 2702.83 2558.163 A 10 2225.17 2138.25 2085.01 2149.476667 2004.81 A 5 1507.55 1505.67 1501.48 1504.9 1360.233 A 2 1389.4 1392.23 1394.7 1392.11 1247.443 A 1 1386.96 1384.98 1382.04 1384.66 1239.993 A 0 1339.47 1339.49 1339.46 1339.473333 1194.807 A Sample is neutralized with HCl to attain desired base concentration. B Corrected values were obtained by subtracting the mean absorbance (144.67) of 0 mg/mL protein to the actual fluorescence intensity means.

2. Percent Recovery

hermostability test Supplementary Table 7. Percent recovery after removal of heat treatment Temperature Recovered % A (C) RFU Recovery 35 45 55 65 75 85 95

7984.547 6762.913 5502.227 4329.413 3042.093 1275.96 116.3667

71.4843 60.54723 49.26051 38.76051 27.23535 11.42346 1.041811

The initial fluorescence intensity used to calculate is 11169.65 RFU.

Stability test in acidic conditions

Stability test in basic conditions