Application of Ratiometric Measurements and Microplate Fluorimetry

Laboratory Experiment pubs.acs.org/jchemeduc

Application of Ratiometric Measurements and Microplate Fluorimetry to Protein Denaturation: An Experiment for Analytical and Biochemistry Students Joaquim T. Marquês and Rodrigo F. M. de Almeida* Centro de Química e Bioquímica, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal S Supporting Information *

ABSTRACT: The number of applications of fluorescence spectroscopy in different areas of chemistry has increased dramatically, in part because a variety of instruments are used to measure fluorescence, including high-throughput microplate readers. Therefore, it is important to introduce students to different instruments. With many instruments, several experimental limitations hamper quantitative treatment of data, unless ratiometric measurements, that is, the ratio of intensity at two different excitation or emission wavelengths, are made. However, such methods are not always applicable. The denaturation of proteins often induces a red-shift of the tryptophan residues emission. Such a shift permits the use of ratiometric measures to obtain the fraction of native and denatured protein. To our knowledge, the use of ratiometric analysis with fluorescence measurements obtained from a microplate reader for the study of protein (biomolecular) denaturation has not been applied as a teaching exercise. In this experiment, the denaturation of hen egg-white lysozyme by guanidine hydrochloride is studied. Students perform ratiometric and singlewavelength measurements and obtain thermodynamic parameters for the denaturation process; they also test the reversibility of denaturation. In these studies the advantages of the ratiometric method are highlighted. Students develop analytical skills and, simultaneously, their understanding of the physical-chemical principles behind protein structural changes. KEYWORDS: Second-Year Undergraduate, Upper Division Undergraduate, Analytical Chemistry, Biochemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Biophysical Chemistry, Fluorescence Spectroscopy, Proteins/Peptides he development of ratiometric fluorescent probes for different (bio)chemical parameters, such as intracellular Ca2+ or cellular viability, underpins the growing importance of understanding the principles behind ratiometric measurements.1,2 However, it is difficult to find experiments involving such measurements that are easily adaptable to the restrictions of a teaching laboratory class and have direct biochemical significance. A recent paper highlighted the advantages of performing many laboratory class experiments with microplate readers.3 In the same paper, the major advantages and limitations of those instruments were also emphasized. As detailed here, ratiometric measurements can be highly desirable to overcome those limitations. This is of special importance because the use of microplate readers is becoming routine in undergraduate laboratory courses.4−9 Ratiometric fluorescence measurements are possible when spectral shifts occur and therefore are suitable to study the exposure of tryptophan residues in proteins upon denaturation using their fluorescence emission. To the best of our knowledge, there are no reports on the application of a ratiometric method to the fluorimetric analysis of protein denaturation for laboratory classes. The experiment described avoids the need for cell culture facilities and expensive ratiometric dyes commonly used to determine intracellular

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© 2013 American Chemical Society and Division of Chemical Education, Inc.

biochemical and clinical parameters, but provides students with the tools they will need to understand, apply, and develop ratiometric assays in their professional careers. Recently, in this Journal experiments were described where the thermal denaturation of lysozyme was followed by differential scanning calorimetry and UV-absorption spectroscopy.10,11 Here, an experiment is described where the denaturation of hen egg-white lysozyme (E.C. 3.2.1.17), a low-cost commercially available protein commonly used in laboratory classes,10,12−15 is studied using a microplate reader, and data is analyzed with a ratiometric method and a single-wavelength method for comparison. The work is suitable for intermediate to upper-division undergraduate students in the subjects of analytical (bio)chemistry, (bio)physical chemistry, and spectroscopy, or to transdisciplinary experiments.16 With this experiment, second-year undergraduate students learn how to optimize a spectroscopic analytical method and, in particular, understand the potentials and limitations of fluorimetry, and why and how many limitations are overcome by using a ratiometric method, using a biochemical application. Other goals are to understand spectral shifts and analysis of protein structural stability. For master (advanced undergraduate) Published: October 7, 2013 1522

dx.doi.org/10.1021/ed300599d | J. Chem. Educ. 2013, 90, 1522−1527

Journal of Chemical Education

Laboratory Experiment

Figure 1. (A) Emission spectra of lysozyme in Tris buffer (pH 7.0) at 24 °C in the presence of the indicated GndHCl concentrations after blank subtraction: (B) normalized to the maximum fluorescence intensity (Imax = 1); (C) an example of a raw emission spectrum and the respective blank. The spectra are representative results obtained by second-year undergraduate students.

If temperature control is available, data at different temperatures can be readily obtained and a complete thermodynamic analysis can be performed.20 The reversibility of the process is tested by diluting one of the samples containing higher denaturant concentration.

students, this experiment is performed as part of a larger project. In addition to the goals described above, additional objectives are to realize that fluorescence spectroscopy encompasses a range of different techniques and to integrate the complementary information obtained into a detailed description of lysozyme structure and dynamics in solution.

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BACKGROUND The study of a protein’s denaturation from the shift of its fluorescence emission can be performed with a large variety of proteins, such as lysozyme,17 provided they contain tryptophan residue(s) in their more hydrophobic interior that become water-exposed upon denaturation. The general advantages of this type of experiment are simplicity, low cost, and illustration of several concepts related to protein denaturation and polarity, including the possibility of quantitative treatment of data and thermodynamic analysis. Moreover, lysozyme denaturation by guanidine hydrochloride (GndHCl) is well described by a twostate model.18 There are two major limitations of the experiments usually proposed: (1) The quantitative treatment is only approximate, unless the data is corrected for light absorption by the denaturant, such as described for the quencher’s absorption in protein quenching experiments;19 however, this correction relies on UV absorbance measurements, which are time-consuming and will add error to the results. (2) The use of a spectrofluorimeter, which is instrumentation that students are not usually familiar with and that poses a practical problem for a class because only one sample can be measured at a time; therefore, most time in a class can be turned into waiting time.3 In the alternative experiment described here, data are collected in a high-throughput microplate fluorescence reader where the reading is much faster and a ratiometric method of data analysis is used that eliminates single-wavelength artifacts.

EXPERIMENTAL PROCEDURE

Students prepare a Tris buffer solution, a standard solution of guanidine hydrochloride in Tris buffer, and a standard solution of hen egg-white lysozyme in Tris buffer. For the denaturation study, a series of varying guanidine hydrochloride concentrations (0−7 M) containing a fixed concentration of lysozyme in Tris buffer are prepared, as well as an identical series of solutions without lysozyme to serve as blanks. A fixed quantity of each solution is added to separate wells of a microplate. The emission spectrum between 310 and 450 nm with excitation at 290 nm is recorded for each solution using a microplate fluorescence reader. For the protein renaturation study, an aliquot of the samples prepared in 6 M guanidine hydrochloride is diluted 3-fold with Tris buffer, and the emission spectra are recorded as before. The data are analyzed by subtracting the blank from the emission spectra and plotting the resulting spectra. From these data, a denaturation curve is prepared using a ratiometric method, and for comparison purposes by a single wavelength method, to obtain a denaturation constant from which the cooperativity of the denaturation process and the free energy of denaturation in water are calculated. The reversibility of the denaturation process is assessed from a comparison of the mole fraction of denatured protein. The details of the procedure are given in the Supporting Information and allow for a comparison with literature results.18 1523




Figure 2. Typical denaturation curves of hen egg-white lysozyme by GndHCl in Tris buffer (pH 7.0) at 24 °C: (A) obtained by undergraduate students using a microplate reader with the ratiometric method; (B) obtained by undergraduate students using a microplate reader with the singlewavelength method; (C) obtained by master students using a conventional fluorimeter with the ratiometric method; (D) obtained by master students using a conventional fluorimeter with the single-wavelength method (the data were corrected for inner filter effect19).

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Emission Spectra and Wavelength Selection

HAZARDS GndHCl and Tris are irritant agents in case of skin and eye contact. GndHCl is hazardous if ingested or inhaled. Hydrochloric acid is very hazardous if ingested or in case of skin and eye contact, it is corrosive and an irritant if it contacts skin or eyes; avoid inhalation. No special care is needed when handling lysozyme.

Emission spectra for lysozyme, after subtraction of the blank, obtained by second-year undergraduate students are shown in Figure 1A; fluorescence intensity increased as the concentration of guanidium hydrochloride increased. The emission spectra normalized to a maximum intensity (Imax = 1) are shown in Figure 1B for the sample without denaturant and at maximum denaturant concentration. This representation was more appropriate to visualize solvatochromic shifts. A typical bathochromic shift in fluorescence emission was observed from an increased polarity of the environment surrounding side-chains of Trp residues. From an analytical point of view, the spectra were very important. Intuitively, a wavelength corresponding to the maximum emission of one of the forms (fully native or fully denatured) of the protein should be used to make a denaturation curve using a single-wavelength method; the two wavelengths for the maximum emission of the native protein and the denatured protein should be used in the ratiometric method. However, due to the simultaneous occurrence of a red shift and a quantum yield increase, and the fact that the emission band is very broad, the fluorescence intensity variations around the maximum are only modest. One wavelength should be selected to have the largest difference between native and denatured samples.21 Therefore, an emission wavelength of 375 nm was used for the single-

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RESULTS The experiment was performed by biochemistry master (advanced undergraduate) classes in 2010 and 2011 (45 students with a 3-year graduation in chemistry or biochemistry), and by a second-year undergraduate class of biochemistry (15 students) in 2012. The experiments were also conducted using conventional spectrofluorimeters by biochemistry master classes in 2008 and 2009. A 3-h period was required for the undergraduate lab and the 15 students were divided into three groups. The ratiometric method was advantageous using a conventional spectrofluorimeter because it eliminated errors that arose from protein concentration variations from sample to sample and solvent changes upon denaturant addition, which are major sources of error regardless of the instrumentation. However, if a microplate reader is not available, the experiment can be performed and used to compare the ratiometric and the singlewavelength methods using a conventional fluorimeter. 1524




0H O

Table 1. Values of ΔGD 2 , m, and c1/2 from Eight Denaturation Curves Obtained from Undergraduate and Master Students and Fraction of Denatured Protein, XD′, Obtained by the Students after the Renaturation Assay Source Students Ratiometric Single-wavelength ref 18

0H2O

ΔGD

Underg. 31.6 ± 3.2 17.2 ± 4.1 37.2

/(kJ mol−1) Master 31.9 ± 1.2 19.0 ± 3.5

m/(kJ mol−2 L) Underg. 7.2 ± 2.2 4.4 ± 1.6 7.8

X D′

c1/2/(mol L1)

Master 7.5 ± 1.1 4.9 ± 1.1

Underg. 4.4 ± 0.6 3.9 ± 0.8 4.2

Master 4.3 ± 0.5 3.9 ± 0.7

Underg. 0.06 ± 0.09 0.14 ± 0.12

Master 0.03 ± 0.06 0.19 ± 0.10

2O Figure 3. Estimation of the free energy of denaturation of lysozyme in water, ΔG0H , from the intercept, and cooperativity of the process, m, from D the slope of a linear least-squares fit to the free energy of denaturation of lysozyme by GndHCl at 24 °C as a function of denaturant concentration 2O − m[GndHCl]: (A) ratiometric method, ΔG0D (kJ (illustrative results obtained by undergraduate students) according to the equation ΔG0D = ΔG0H D 0 −1 −1 mol ) = (36.1 ± 2.5) − (8.9 ± 0.6)[GndHCl]; (B) single wavelength method, ΔGD (kJ mol ) = (17.1 ± 1.8) − (3.4 ± 0.4)[GndHCl].

method (Figure 2C), even though, in the curve obtained from single wavelength analysis for data collected with a conventional fluorimeter, the values of fluorescence intensity were corrected for the inner filter effect (Figure 2D). One of the problems associated with the analysis of denaturation curves was that, for the native and denatured states of the protein, the experimental observable, in this case the fluorescence intensity, was dependent on denaturant concentration because the denaturant concentration changed general solvent properties. In single-wavelength analysis, several methods have been proposed that implied either extrapolation of the extremes of the denaturation curve into the denaturing region,22 or a nonlinear analysis of the data.21,23 In the ratiometric curve, this was not necessary as a much more constant value was obtained outside the transition region. The (standard) free energy of denaturation, ΔG0D, for each denaturant concentration was calculated and plots of ΔG0D versus [GndHCl] were made.18 From the linear least-squares fit of a straight line to the data, the slope was a measure of the cooperativity of the denaturation process, m, and the intercept was the free energy of denaturation of lysozyme in water, 2O ΔG0H and reflected the thermodynamic stability of the D protein in solution:18

wavelength method and wavelengths of 375 and 340 nm for the ratiometric method. These wavelengths provided additional advantages from the analytical point of view, because at 375 nm the background was small and using 340 nm instead of 330 or 335 nm, which corresponds to the maximum emission of the native protein, avoided the Raman band of water, which minimized errors that arose from the blank subtraction. It was important to plot the spectra of the blank and the respective sample (Figure 1C was made for the sample with 3.8 M denaturant) and to discuss these aspects of the experimental design with students. If students were not told to plot a blank spectrum, they often plotted only the final result after the subtraction of the blank and, thus, were not aware of the importance of the background to the final result and the optimization of the analytical method. Denaturation Curves

The purpose of obtaining a spectroscopic signal as a function of denaturant concentration was to calculate the molar fractions of denatured enzyme, XD. From these values, the free energy of denaturation was calculated via the denaturation constant. The denaturation curves obtained from the ratiometric and singlewavelength methods by second-year undergraduate students using a microplate reader are shown in Figure 2, panels A and B, respectively. Comparing the two curves, data scattering was considerably reduced using the ratiometric method. The denaturant concentration necessary to achieve 50% of denaturation, c1/2, was read directly from these curves. In both cases, a value of ∼4 M, close to that previously reported (4.2 M),18 was obtained. The values of c1/2 were also calculated from the thermodynamics analysis of the curves described below (Table 1). The curves obtained by master students using a conventional fluorimeter are shown in Figure 2C,D. As in the case of a microplate reader, a clear improvement of the curve was obtained when the data were analyzed by the ratiometric

ΔG D0 = ΔG D0H2O − m[GndHCl]

The value of c1/2 was also calculated from these parameters as ΔG D0H 2 O /m, because it corresponds to the denaturant concentration at which ΔG0D equals zero. The calculation was performed for both ratiometric and single-wavelength methods (Figure 3), and the results are shown in Table 1, together with data from the literature.18 In this reference, the fraction of denatured protein was calculated from changes in molar absorption coefficient at 292 nm; therefore, they do not reflect exactly the same structural changes as the ones reported here 1525




renaturation study. This material is available via the Internet at http://pubs.acs.org.

and may yield slightly different values for the denaturation parameters.24 The ratiometric method yielded parameters in closer agreement with the literature and with reduced uncertainty. These values are typical of globular proteins close to room temperature (25 ± 2 °C) with a denaturation curve well described by a two-state model, including lysozyme, ribonuclease A, α-lactalbumin, and myoglobin,18,20,23 and azurin from Pseudomonas aeruginosa, which was studied using a fluorescence ratiometric method.25 In these examples, values of the free energy of denaturation in water, which is independent of the denaturing agent, ranged from 16.7 to 40.3 kJ mol−1; for the other parameters, values for denaturation by GndHCl were in the ranges of 5.3−25.5 kJ mol−2 L for m and 1.5−4.2 mol L−1 for c1/2.18,20,23,25

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Corresponding Author

*E-mail: [email protected], [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financed by Portuguese national funds through FCT, the Portuguese Foundation for Science and Technology, through “Ciência 2007” and “Investigador FCT 2012” (POPH, Fundo Social Europeu) Initiatives, PEst-OE/QUI/UI0612/ 2011-2013 and SFRH/BD/64442/2009. The authors thank a reviewer for the thorough and helpful comments provided for the paper.

Renaturation

The fraction of denatured protein, XD′, in the renatured sample was calculated, and it was concluded that lysozyme denaturation by GndHCl was a reversible process on the time scale of minutes or less, as a value of denatured fraction similar to one previously obtained for 2 M GndHCl (Figure 2B) was recovered (Table 1). Interestingly, in the calculation by the single-wavelength method, the dilution of the protein had to be taken into account (after blank subtraction the fluorescence intensity had to be multiplied by the dilution factor) and the error in the renatured fractions increased up to several fold. This was not the case with the ratiometric calculation, where no dilution had to be considered as this kind of measurement was independent of total fluorophore concentration, further illustrating the usefulness of this type of fluorimetric assay.

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REFERENCES

(1) Valeur, B.; Berberan-Santos, M. N. Molecular Fluorescence: Principles and Applications; 2nd ed.; Wiley VCH: Weinheim, 2012; pp 3−72, 265−276, 462−466. (2) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; 3rd ed.; Springer: New York, 2006; pp 1−60, 530−573. (3) Botasini, S.; Luzuriaga, L.; Cerda, M. F.; Mendez, E.; FerrerSueta, G.; Denicola, A. Multiple experiments and a single measurement: Introducing microplate readers in the laboratory. J. Chem. Educ. 2010, 87, 1011−1014. (4) Craig, P. A. A project-oriented biochemistry laboratory course. J. Chem. Educ. 1999, 76, 1130−1135. (5) Bevilacqua, V. L. H.; Powers, J. L.; Vogelien, D. L.; Rascati, R. J.; Hall, M.; Diehl, K.; Tran, C.; Jain, S. S.; Chabayta, R. Collaboration between chemistry and biology to introduce spectroscopy, electrophoresis, and molecular biology as tools for biochemistry. J. Chem. Educ. 2002, 79, 1311−1313. (6) Wentland, M. P.; Raza, S.; Gao, Y. 96-well plate colorimetric assay for Ki determination of (±)-2-benzylsuccinic acid, an inhibitor of carboxypeptidase a: A laboratory experiment in drug discovery. J. Chem. Educ. 2004, 81, 398−400. (7) Taylor, A. T. S. Screening a library of household substances for inhibitors of phosphatases: An introduction to high-throughput screening. Biochem. Mol. Biol. Educ. 2005, 33, 16−21. (8) Powers, J. L.; Kiesman, N. E.; Connie, M. T.; John, H. B.; Bevilacqua, V. L. H. Lactate dehydrogenase kinetics and inhibition using a microplate reader. Biochem. Mol. Biol. Educ. 2007, 35, 287− 292. (9) Goyette, S. R.; DeLuca, J. A semester-long student-directed research project involving enzyme immunoassay: Appropriate for immunology, endocrinology, or neuroscience course. CBE Life Sci. Educ. 2007, 6, 332−342. (10) Schwinefus, J. J.; Schaefle, N. J.; Muth, G. W.; Miessler, G. L.; Clark, C. A. Lysozyme thermal denaturation and self-interaction: Four integrated thermodynamic experiments for the physical chemistry laboratory. J. Chem. Educ. 2008, 85, 117−120. (11) Schwinefus, J. J.; Leslie, E. J.; Nordstrom, A. R. The effect of ethylene glycol, glycine betaine, and urea on lysozyme thermal stability. J. Chem. Educ. 2010, 87, 1393−1395. (12) Garrett, E.; Wehr, A.; Hedge, R.; Roberts, D. L.; Roberts, J. R. A novel and innovative biochemistry laboratory: Crystal growth of hen egg white lysozyme. J. Chem. Educ. 2002, 79, 366−368. (13) Peterson, R. R.; Cox, J. R. Integrating computational chemistry into a project-oriented biochemistry laboratory experience: A new twist on the lysozyme experiment. J. Chem. Educ. 2001, 78, 1551− 1555.

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SUMMARY This experiment was used to develop critical thinking by detecting the major sources of error in a given analytical method and ways to overcome them. For second-year undergraduate students of biochemistry, the experiment was performed with more support during the class, and “dead times” were used to ensure that the rationale behind the experiment and the major concepts were well understood by students. The main steps of data analysis were done in class using results obtained by the students after the experimental part was finished. For master students in biochemistry, this experiment was integrated into a project concerning the structure and dynamics of lysozyme in solution. A minimal amount of information was given, and each group wrote a report in the form of a scientific article. The undergraduate students delivered individual reports, and for both undergraduate and master students, there was a final oral examination, which included the other experiments of the laboratory course. The students were positively assessed in this experiment, presenting a higher degree of success than with experiments that had been used for several years in the laboratory courses. The range of experimental results obtained is given in Table 1.

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AUTHOR INFORMATION

ASSOCIATED CONTENT

* Supporting Information S

Student handout; notes for instructors, including other possible extensions, adaptations, and main problems that might be faced. It is also explained how to optimize the use of a 96-well plate for running a complete replicate of the denaturation/ 1526




(14) Hall, M. L.; Guth, C. A.; Kohler, S. J.; Wolfson, A. J. Advanced instrumentation projects for first-year biochemistry laboratory. Biochem. Mol. Biol. Educ. 2003, 31, 115−118. (15) Kurtin, W. E.; Lee, J. M. The free energy of denaturation of lysozyme: An undergraduate experiment in biophysical chemistry. Biochem. Mol. Biol. Educ. 2002, 30, 244−247. (16) Farinha, C. M.; Freire, A. P. Teaching biochemistry at Lisbon UniversityFacing the challenge of the Bologna Declaration in the 25th anniversary of the biochemistry course. Biochem. Mol. Biol. Educ. 2007, 35, 422−424. (17) Imoto, T.; Rupley, J. A.; Tanaka, F.; Forster, L. S. Fluorescence of lysozyme: Emissions from tryptophan residues 62 and 108 and energy migration. Proc. Natl. Acad. Sci. U.S.A. 1972, 69, 1151−1155. (18) Ahmad, F.; Bigelow, C. C. Estimation of the free energy of stabilization of ribonuclease A, lysozyme, alpha-lactalbumin, and myoglobin. J. Biol. Chem. 1982, 257, 2935−2938. (19) Coutinho, A.; Prieto, M. Ribonuclease T1 and alcohol dehydrogenase fluorescence quenching by acrylamide: A laboratory experiment for undergraduate students. J. Chem. Educ. 1993, 70, 425− 428. (20) Silverstein, T. P.; Blomberg, L. E. Probing denaturation by simultaneously monitoring residual enzyme activity and intrinsic fluorescence: An undergraduate biochemistry experiment. J. Chem. Educ. 1992, 69, 852−855. (21) Walters, J.; Milam, S. L.; Clark, A. C. Practical approaches to protein folding and assembly: Spectroscopic strategies in thermodynamics and kinetics. Methods Enzymol. 2009, 455, 1−39. (22) Pace, C. N. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 1986, 131, 266− 280. (23) Jones, C. M. An introduction to research in protein folding for undergraduates. J. Chem. Educ. 1997, 74, 1306−1310. (24) Kuramitsu, S.; Hamaguchi, K. Difference absorption spectra, circular dichroism, and disulfide cleavage of hen and turkey lysozymes in the alkaline pH region. J. Biochem. 1979, 85, 443−456. (25) Strambini, G. B.; Gonelli, M. Protein stability in ice. Biophys. J. 2007, 92, 2131−2138.

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Application of Ratiometric Measurements and Microplate Fluorimetry

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