Determination of GFP Chromophore pka, and EGFP Concentration via BCA & Bradford Assays and UV Absorbance By: Aaron Coffey

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1 Determination of GFP Chromophore pka, and EGFP Concentration via BCA & Bradford Assays and UV Absorbance By: Aaron Coffey Abstract Concentrations of EGFP for two pools of purified EGFP were calculated with several different assays including BCA, Bradford, and UV absorption and found to range from mg/ml and mg/ml. Percentage of EGFP chromophore formation was found to be 31.6%. The pka of this chromophore was estimated at 5.50 via a titration analogous method. Introduction The EGFP solutions resulting from Ni-affinity and anion-exchange chromatographies were evaluated using three different methods of analysis. Specifically, these three tests helped in calculating the concentration of EGFP in the final purification products. The techniques include a Bicinchoninic Acid 1 (BCA) assay, a Bradford assay, and a UV absorbance scan. The other investigation performed was an inquiry as to the pka of the subject GFP s chromophore. This was executed by way of a method analogous to titration. The BCA test exploits a two-part reaction among its three necessary reagents protein, copper(ii), and bicinchoninic acid. 1 First, the Cu 2+ ions (usually originating from aqueous CuSO 4 ) are reduced to Cu + by the functional groups in peptide bonds, tyrosine, tryptophan, and to a lesser extent cystine, cysteine, and histidine residues. 1 Then, the BCA forms an intense purple complex with [the] cuprous ions, 1 the A 560 of which can be plotted against the concentration of protein in the solution to realize a trendline and equation. Similar to DNA ladders or MW markers and their use in approximating sizes of molecules, these standard 1

2 concentrations and the curve they produce can be used as a guide in comparing and estimating the concentrations of molecules. This method was first called a Lowry assay, which used the Folin Ciocalteu reagent (phosphomolybdate and phos-photungstate) 1 instead of BCA; BCA is now preferred because it is less sensitive to compounds that can interfere and cause misrepresentations of protein concentration. 1 The Bradford assay is similar to the BCA analysis in that it also involves a molecule whose absorption spectrum shifts upon interacting with protein. However, instead of requiring two steps, the Bradford reagent (Coo-massie Brilliant Blue G-250) 1 simply binds to arginine, histidine, phenylalanine, tryptophan and tyrosine residues, and hydrophobic interactions 1 in acidic environments, causing a metachromatic shift from 465 to 595 nm due to stabilization of the anionic form of the dye. 1 The A 595 is used in this test and is also plotted against protein concentration for the purpose of estimating protein concentration. Absorption of UV radiation was another process used to determine the protein concentration in the two samples. The data gathered from an absorption scan can be entered into Beer s law to calculate sample concentration. Beer s law can be written as: Equation 1: A ε c l where A=absorbance (also written as ln ), ε=extinction coefficient (M -1 cm -1 ), [c]=concentration (M), and l=distance light traveled through sample (cm). A molecule s extinction coefficient represents its ability to absorb photons; it can also be described as the size a molecule appears to be to a photon. The concentration of matured GFP chromophore was also found this way after denaturing the protein with NaOH. 2

3 In addition to quantifying the GFP in the samples, the protein s chromophore was also examined. This chromophore can be ionized by deprotonating the hydroxyl of its phenol group. The neutral form has an absorption peak at 400nm, and the deprotonated form has an absorption peak at 489nm. 2 The purpose of this phase was to determine at what ph said deprotonation will occur. Similar to titration, samples of GFP were placed in solutions with increasing ph; each mixture was scanned for A The A 489 for each solution was plotted against the ph values, and the resulting graph was used to estimate the pka of the chromophore. From this graph the pka was calculated using the Henderson-Hasselbalch equation, written as Equation 2: ph pk log where [A - ] is the concentration of conjugate base, and [HA] is the concentration of the original acid. Methods BCA and Bradford Assays The first phase began with the creation of a control group which consisted of the protein bovine serum albumin (BSA). To one row of a 96-well plate the following volumes of water were added to the corresponding well (#): (1)250µl, (2)80µl, (3)65µl, (4)50µl, (5)40µl, (6)40µl, (7)30µl, and (8)0µl. 275µl BSA solution (2.0mg/ml) was added to the 8 th well (the well with 0µl water). Dilutions of the BSA were made by transferring volumes between wells in the following order: 220µl (8) to (7), 210µl (7) to (6), 210µl (6) to (5), 200µl (5) to (4), 185µl (4) to (3), and 170µl (3) to (2). 3

4 Into the wells of two other rows on the plate were placed 10µl aliquots from the corresponding wells of diluted standards described above. 10µl from the post nickel column pool were placed in each of the first three wells of the row below these; and 10µl from the post DEAE column pool were put in the next three wells of this row; 10µl water was added to the 7 th well as a blank. 240µl BCA reagent (50 parts pre-mixed bicinchoninic acid, 1 part 4% CuSO 4 5H 2 O) were added to the first eight wells of the three rows being used above. The plate was then covered with Parafilm and kept at 37 C for 30min. An A 560 scan of the plate was taken with a BioTek Synergy HT after incubation. This concluded the BCA test. For the Bradford assay, 5µl aliquots of each of the eight protein standard dilutions were transferred to corresponding wells in two rows on another plate. 5µl from the post nickel column pool were placed in each of the first three wells of the row below these; and 5µl from the post DEAE column pool were put in the next three wells of this row; 5µl water was added to the 7 th well as a blank. 245µl Bradford reagent (Coo-massie Brilliant Blue G-250) were added to each of these three rows at 30 second intervals. The plate was left at room temperature for about 5min, then scanned for A 595 with the BioTek Synergy HT. Optical Properties and Chromophore pka To test the optical properties of GFP under different conditions, another 96-well plate was used to house samples for absorbance scanning. 250µl HEPES buffer (50mM HEPES ph7.9, 300mM NaCl) were added to well A2 as a blank, and 50µl post DEAE column purified EGFP with 200µl HEPES buffer were added to A1. 250µl 0.2M NaOH were added to B1 as a blank, and 25µl post DEAE column purified EGFP with 225µl 0.2M NaOH were added to B2. 250µl urea buffer (8.0M urea, 50mM HEPES ph 7.9) were added to C2 as a blank, and 25µl post DEAE column purified EGFP with 225µl urea buffer were added to C1 after being heated 4

5 to 95 C for 5min. The plate was scanned for A for the row with HEPES and A for the rows with NaOH and urea with a BioTek Synergy HT. For fluorescence emission testing, 1.5ml HEPES (50mM HEPES ph7.9, 300mM NaCl) and 50µl post DEAE column purified EGFP were transferred to a cuvette. The mixture was excited with 455nm radiation and scanned for fluorescence from nm with a Shimadzu 1501 Spectrofluorophotometer. The final investigation was meant to find the pka of the GFP chromophore. Instead of titration of a sample of the protein, another 96-well plate was used with various ph solutions. 50µl purified EGFP were placed into each of wells A1-A7. 200µl of the following solutions were added to the indicated wells: (A1)pH 4.00, 75mM acetate, 140mM NaCl; (A2)pH 5.07, 75mM acetate, 140mM NaCl; (A3)pH 5.40, 75mM acetate, 140mM NaCl; (A4)pH 5.74, 75mM acetate, 140mM NaCl; (A5)pH 6.40, 75mM MES buffer, 140mM NaCl; (A6)pH 6.93, 75mM Bis-Tris, 140mM NaCl; (A7)pH 7.73, 75mM HEPES, 140mM NaCl. The plate was scanned for A with a BioTek Synergy HT. Results The results gathered during week one included the standard curves from the BCA and Bradford assays and the estimated EGFP concentrations calculated from each. The BCA data will be examined first. 5

6 A 560 vs. Protein Concentration Absorption of 560nm Light y = x R² = BSA Standard Concentration (mg/ml) Figure 1: Curve and trendline of BSA standard concentrations for BCA assays plotted against their A 560. Trendline equation used to estimate [EGFP] in pools from post Nickel-affinity and post anion-exchange chromatography columns. Based on the average absorptions of the three samples taken from each column s pool, the trendline equation in Figure 1 was used to calculate the post-ni pool s [EGFP] to be 2.134mg/ml and the post-deae pool s [EGFP] to be 2.729mg/ml. This was computed in the following manner: y x A x mg ml x x. 6

7 The data from the Bradford assay was compiled with the same approach, seen below A 595 vs. Protein Concentration ABsorption of 595nm Light y = x R² = BSA Standard Concentration (mg/ml) Figure 2: Curve and trendline of BSA standard concentrations for Bradford assays plotted against their A 595. Trendline equation used to estimate [EGFP] in pools from post Nickel-affinity and post anion-exchange chromatography columns. Calculated as above, the Bradford test data generated values of 1.977mg/ml for [EGFP] in the post-ni column pool and 2.126mg/ml for [EGFP] in the post-deae pool. The second phase involved other methods of determining protein concentration including UV absorption scans of the protein in solution with HEPES buffer and NaOH. The scans corresponding graphs can be viewed below. 7

8 Absorbance Absorbance vs. Wavelength Wavelength (nm) EGFP HEPES Buffer Figure 3: A scan of two wells containing 250µl HEPES buffer (50mM HEPES ph7.9, 300mM NaCl) and 50µl post DEAE column purified EGFP with 200µl HEPES buffer. Two major peaks in the EGFP absorption data lie around 280nm and 490nm, with a very minor shoulder peak near 400nm. Beer s law (Equation 1) was employed to calculate the concentration of EGFP in the sample used to create Figure 3. The concentration ([c]) had to be increased five-fold due to its five-fold dilution in the well. The molecular weight of GFP with the histidine tag (31,079g/mol) was also utilized to obtain a value in g/l. As mentioned in previous experiments, all peptide bonds optimally absorb 280nm light; the absorption value in this region of Figure 3 was used in the following calculation. The extinction coefficient (ε 280 =21,050 M -1 cm -1 ) 2 was taken from relevant literature on GFP. A ε c l ,050 M cm c 1cm c M; 5 c M mol ,079 g L mol.. 8

9 Since the total volume of the post-anion-exchange column pool was 8ml, the total weight of EGFP in this pool can be found by: mg 8ml. ml A similar test was run with 0.2M NaOH, and its results yielded the following graph. Absorbance vs. Wavelength Absorbance Wavelength (nm) EGFP NaOH Because the GFP chromophore becomes denatured in basic conditions, the concentration of the chromophore itself can be discovered with the help of Beer s law and the extinction coefficient λ max = 37,000 M -1 cm -1 ) 2. Beer s law produced a chromophore concentration of M in the post-deae pool. This means that the percent chromophore in that pool was 31.6%. Figure 4: A scan of two wells containing 250µl 0.2M NaOH and 25µl post DEAE column purified EGFP with 225µl 0.2M NaOH. Absorbance maximum resides at 446nm. A similar test was run with 8M urea, and its results yielded the following graph. It is important to note that the solution lost almost all color after being heated with the urea. 9

10 Absorbance Absorbance vs. Wavelength Wavelength (nm) Figure 5: A scan of well containing 25µl post DEAE column purified EGFP with 225µl urea buffer (8.0M urea, 50mM HEPES ph 7.9). Two absorbance maxima are visible at 384nm and 458nm. A fluorescence scan of the post-deae pool was also taken, and its data formed this plot Fluorescence vs. Wavelength Fluorescence Wavelength (nm) Figure 6: Emission scan from nm with excitation at 455nm of a cuvette containing 1.5ml HEPES (50mM HEPES ph7.9, 300mM NaCl) and 50µl post DEAE column purified EGFP. Large emission plateau ranges from nm. 10

11 The final inquiry of this experiment was in regard to the pka of the EGFP chromophore. A scans of the molecule in several different ph levels resulted in the following chart. 0.6 Absorbance vs. ph Absorbance A400 of EGFP A490 of EGFP ph Figure 7: A 400 and A 490 scans of post-deae column EGFP pool in ph levels: 4.00, 5.07, 5.40, 5.74, 6.40, 6.93, and These plots intersect at about The most significant feature of Figure 7 is the intersection of the two plots at a ph of about With more in-depth knowledge of the EGFP chromophore, and thanks to Equation 2, it can be assumed that its pka lies at the ph corresponding to said intersection. Simply put, at this intersection: neutral EGFP EGFP anion EGFP anion log 0 neutral EGFP which means ph pk 0 pka

12 Discussion With regard to Figures 1 and 2 and the concentration values they helped acquire, it can be said that the BCA and Bradford assays produced results that were quite precise, considering that the largest difference within the two pools was 0.603mg/ml. This is due to the fact that the linear trendline in both graphs had an R 2 value (representing the trendline s accuracy) of over 0.9. Taking only the R 2 number into account, the BCA test seemed to be slightly more accurate than the Bradford method because its number was higher. This could be attributed to the timing of the addition of the Bradford reagent varying slightly among wells, causing the reaction times to not be equal. It was decided that because the His-tag had not been removed from either pool, for the purpose of the experiment, only one pool need be entered in the remaining assessments. The post-deae pool was chosen because it had the most volume, which would allow for tests to be run more than once if necessary. The protein concentration found using Figure 3 was slightly higher than those of the BCA and Bradford assays, though still close enough to be considered precise. Unfortunately, this method does not have an R 2 value to compare with other tests to determine its relative accuracy. The data constructed with Figure 4 revealed that the concentration of actual EGFP chromophore was much less than EGFP. This means that only 31.6% of the EGFP was able to completely form its chromophore. Since O 2 is required for the formation of the chromophore, it is very likely that the EGFP molecules that did not create a chromophore did not have an adequate supply of oxygen. 3 Also, the spontaneity of the chromophore s formation and complete lack of assistance from any enzymes greatly decreases the likelihood of creation. 3 12

13 The chemical denaturation of the EGFP chromophore with urea, illustrated in Figure 5, still rendered noticeable absorption peaks, indicating the presence of the chromophore. In comparison to Figure 4, the absorption peaks in Figure 5 are significantly shifted to the left of the graph, absorbing higher energy radiation. If the His-tag had been removed previously, Figure 6 would have shown the difference in fluorescence peaks between the His-tag pools. It greatly emphasizes the +His-tag chromophore s ability to emit wavelengths of nm. The chromophore s pka was estimated using Figure 7. Only A 400 and A 490 are shown here because there are the absorption maxima of the molecule in the neutral and anionic state. It can also be assumed that when these lines intersect the concentrations of each form are equal because they are equally absorbing their optimal wavelength; thus, the ph at which this occurs is also the molecule s pka References 1 Noble, J.E., and Bailey, M.J.A. (2009). Methods in Enzymology. 463, /S (09) Lefler, S. (2011). BCH467 Lab 10 Protocol Lefler, S. (2011). BCH467 Lab 10 Recitation 13