Facile measurement of protein stability and folding kinetics using a nano differential scanning fluorimeter

Abstract

With advancements in high‐throughput generation of phenotypic data on mutant proteins, it has become important to individually characterize different proteins or their variants rapidly and with minimal sample consumption. We have made use of a nano differential scanning fluorimetric device, from NanoTemper technologies, to rapidly carry out isothermal chemical denaturation and measure folding/unfolding kinetics of proteins and compared these to corresponding data obtained from conventional spectrofluorimetry. We show that using sample volumes 10‐50‐fold lower than with conventional fluorimetric techniques, one can rapidly and accurately measure thermodynamic and kinetic stability, as well as folding/unfolding kinetics. This method also facilitates characterization of proteins that are difficult to express and purify.

Introduction

A protein typically adopts a folded conformation to carry out biological function. Under physiological conditions, the folded state is usually more stable than its corresponding unfolded, inactive state. Protein stability is either thermodynamically or kinetically controlled.1, 2, 3, 4 Widely used techniques for deciphering thermodynamic and kinetic stability of proteins involve measurements of intrinsic fluorescence either at equilibrium or as a function of time.5, 6, 7, 8 Such measurements are sample and labor intensive. This is a significant limitation for unstable proteins with low purification yields. Here we describe the use of a nano differential scanning fluorimeter (nanoDSF) instrument, the Prometheus NT.48 from NanoTemper technologies to overcome these limitations.

nanoDSF is an advanced differential scanning fluorimetry technology that detects small changes in the fluorescence of tryptophan upon folding or unfolding as a function of temperature or other solution conditions. The fluorescence of tryptophan in a protein is strongly dependent on its surroundings.9 By following changes in fluorescence, chemical and thermal stability10, 11 can be assessed in a truly label‐free fashion.

We describe equilibrium, isothermal, unfolding studies for two proteins, homodimeric Escherichia coli CcdB and monomeric hen egg white lysozyme (HEWL) using both a conventional fluorimeter and nanoDSF. We also measure unfolding and refolding kinetics of CcdB using nanoDSF and compare with previously published kinetic data.7, 8 The data demonstrate that nanoDSF can be used to accurately and rapidly measure protein stability and folding/unfolding kinetics with minimal sample consumption.

Results

Biophysical characterization of the proteins

CcdB protein was purified by affinity purification against immobilized ligand CcdA. The purified CcdB and HEWL [Fig. 1(a)] were subjected to thermal denaturation, monitored by intrinsic fluorescence of tryptophan and tyrosine residues as a function of temperature, and the apparent thermal melting temperature (T_m) was calculated [Fig. 1(b)]. The fluorescence of the tryptophan and tyrosine residues in a protein is strongly dependent on the surrounding environment.9 Changes in the structure of the protein have an effect on both the intensity and the emission wavelength, especially of tryptophan fluorescence. The nanoDSF (Prometheus NT.48) is equipped with fluorescence detectors that measure the fluorescence intensity at two different wavelengths, 330 nm and 350 nm, thus being sensitive for both, the change in fluorescence intensity and the shift of the fluorescence maximum upon unfolding. The apparent T_m can be calculated from the changes in tryptophan fluorescence intensity, or from the ratio of tryptophan emission at 330 nm and 350 nm, as a function of temperature. CcdB and HEWL are homodimeric and monomeric proteins, respectively. The oligomeric states of the purified CcdB and HEWL were determined by subjecting ∼120 μg of protein to size exclusion gel chromatography (SEC) using a Superdex‐200 100/300GL [Fig. 1(c)]. The expected and inferred molecular weight from SEC for CcdB is 23.4 kDa and 24 kDa, respectively and for HEWL it is 14.4 kDa and 15 kDa, respectively.

Figure 1.

Biophysical characterization of the proteins used in the study. (a) SDS PAGE profile of CcdB (1) and HEWL (2) are shown. (b) Thermal unfolding traces of CcdB in 10 mM HEPES, CcdB in 200 mM HEPES, and HEWL are shown in black, red, and blue respectively. (c) Confirmation of oligomeric state of CcdB and HEWL using size exclusion chromatography (SEC) in PBS at 4 °C are shown in red and blue, respectively.

Isothermal denaturation

Equilibrium unfolding experiments for both CcdB and HEWL were carried out by conventional fluorimetry and with a nanoDSF (Prometheus NT.48). The data were fitted to N₂ → 2D and N → D unfolding models for homodimeric CcdB and monomeric HEWL, respectively (Figs. 2, 3, Table 1). The raw data of CcdB (10 mM HEPES, pH 8.4) and HEWL (PBS, pH 7.4) in presence of GdnCl and urea is also plotted in terms of 350/330 ratio as a function of denaturant concentration (Fig. 4). A repeat experiment with a different set of capillaries demonstrated reproducibility [Fig. 5(a)]. We confirmed that the minimum concentration of protein that can be reliably used for nanoDSF is 15 μg/mL, as suggested by the manufacturer [Fig. 5(b)]. We choose these proteins because they represent different unfolding models and both have been extensively characterized previously.7, 12, 13, 14, 15, 16, 17 The midpoint of chemical denaturation (C_m), ΔG⁰, and m‐values measured is similar in both the instruments. The C_m, ΔG⁰, and m‐values of HEWL measured is similar to that reported earlier.18

Figure 2.

GdnCl denaturation of CcdB under two different buffer conditions. Equilibrium denaturation profiles of CcdB in 10 mM HEPES, (•) pH 8.4 and 200 mM HEPES (▾), pH 8.4 at 25 °C using both fluorimeter and nanoDSF. Isothermal melts were carried out using 47 μg/mL (2 μM) protein in both cases. The experimental data are shown in dots or inverted triangles, while the fits are shown as smooth lines. The theoretical curves were obtained by fitting all the melts with two‐state unfolding models (N₂↔2D) as described previously.7

Figure 3.

GdnCl denaturation of HEWL in the presence of two different urea concentrations. Equilibrium denaturation profiles of HEWL at PBS, pH 7.5 in the presence of 1 (▾) and 2 M (•) urea at 25 °C using both fluorimeter and nanoDSF. The protein concentration used for isothermal melts was 15 μg/mL (1.05 μM). The experimental data are shown in dots or inverted triangles, while the fits are shown in smooth lines. The theoretical curves were obtained by fitting all the melts with a two‐state unfolding model (N↔D) as described previously.13

Table 1.

Thermodynamic Parameters of CcdB and Lysozyme Measured under Different Buffer Conditions^a

Proteins	Buffer	Fluorimeter			nanoDSF
		ΔG0 (kcal.mol−1)	m (kcal.mol−1 M−1)	Cm (M)	ΔG0 (kcal.mol−1)	m (kcal.mol−1 M−1)	Cm (M)
CcdB	10 mM HEPES, pH 8.4	22.2 ± 0.08	4.89 ± 0.04	2.49 ± 0.05	22.4 ± 0.09	4.94 ± 0.06	2.51 ± 0.02
CcdB	200 mM HEPES, pH 8.4	23.7 ± 0.07	4.60 ± 0.05	3.03 ± 0.05	23.9 ± 0.05	4.48 ± 0.06	3.2 ± 0.1
Lysozyme	1 M Urea, PBS, pH 7.5	7.7 ± 0.07	2.80 ± 0.05	3.33 ± 0.05	7.5 ± 0.05	2.67 ± 0.06	3.35 ± 0.1
Lysozyme	2 M Urea, PBS, pH 7.5	6.9 ± 0.05	2.10 ± 0.05	2.94 ± 0.05	7.1 ± 0.08	2.3 ± 0.06	2.93 ± 0.02

^aReported standard errors are derived from two independent experiments.

Figure 4.

GdnCl and urea denaturation of CcdB and HEWL using 48 different denaturant concentrations. Raw equilibrium denaturation profiles of CcdB in 10 mM HEPES, pH 8.4 (a‐b) and HEWL (c‐d) at PBS, pH 7.5 using GdnCl () and urea (), at 25 °C using nanoDSF. Data is plotted in terms of ratio of fluorescence intensity at 350/330 nm as a function of denaturant concentration. Isothermal melts were carried out using 47 μg/mL (2 μM) for CcdB and 15 μg/mL (1.05 μM) for HEWL. The experimental data are shown in dots, while the fits are shown as smooth lines. The theoretical curves were obtained by fitting all the melts with two‐state unfolding models (N↔D for HEWL and N₂↔2D for CcdB) as described previously.7, 13

Figure 5.

Measurement reproducibility and determination of sample sensitivity of nanoDSF using two different sets of capillaries for 48 different denaturant concentrations. Equilibrium GdnCl denaturation profiles of two different concentration of HEWL (a) 15 μg/mL and (b) 7.5 μg/mL in PBS, pH 7.5 at 25 °C using nanoDSF. Two set of capillaries [capillary 1 () and capillary 2 ()] were used to examine the reproducibility of the data. The data points in denaturation profiles using 7.5 μg/mL of HEWL show increased scatter as compared to data for 15 μg/mL.

Isothermal refolding and unfolding kinetics study

Refolding and unfolding kinetics following manual mixing for CcdB was monitored by time‐course fluorescence spectroscopy as a function of time at 25 °C. Refolding was carried out with 2 μM of purified proteins in either 10 or 200 mM HEPES, at pH 8.4 at a final GdnCl of 1.5 M. Refolding for CcdB occurs with a significant burst phase and a slow phase as observed earlier8 with measured rates similar in both the instruments in both buffer conditions (Fig. 7, Table 2). The unfolding trace of CcdB when fitted to the three‐parameter unfolding equation, gives similar fitted unfolding rates in all conditions (Fig. 6, Table 2). These experiments indicate that the capillary‐based nanoDSF can also be used to make isothermal, time‐dependent, kinetic measurements.

Figure 7.

Refolding kinetic studies of CcdB under different buffer conditions. CcdB exhibits biphasic refolding kinetics with a fast and a slow phase. Kinetic traces at 2 μM protein concentration of (a) CcdB in 10 mM HEPES, pH 8.4 and (b) CcdB in 200 mM HEPES, pH 8.4 using both fluorimeter and nanoDSF are shown. All studies were carried out at 25 °C.

Table 2.

Kinetic Parameters for Refolding and Unfolding of CcdB Measured under Different Buffer Conditions

Buffer/Instrument	Refolding_1.25 M GdnCl
	Fast phase			Slow phase		Unfolding_3M GdnCl
	a0	a1	k1 (s−1)	a2	k2 (s−1)	a0	a1	K1 (s−1)
10 mM HEPES, pH 8.4/Fluorimeter	0.02 ± 0.02	0.7 ± 0.01	0.06 ± 0.005	0.28 ± 0.02	0.001 ± 0.0003	0.85 ± 0.01	0.15 ± 0.02	0.015 ± 0.0001
10 mM HEPES, pH 8.4/nanoDSF	0.02 ± 0.01	0.8 ± 0.01	0.04 ± 0.005	0.18 ± 0.02	0.003 ± 0.0003	0.87 ± 0.04	0.13 ± 0.01	0.020 ± 0.0003
200 mM HEPES, pH 8.4/Fluorimeter	0.02 ± 0.03	0.59 ± 0.02	0.031 ± 0.0005	0.41 ± 0.02	0.001 ± 0.003	0.68 ± 0.02	0.32 ± 0.03	0.006 ± 0.00005
200 mM HEPES, pH 8.4/nanoDSF	0.02 ± 0.02	0.55 ± 0.03	0.027 ± 0.0008	0.43 ± 0.03	0.002 ± 0.001	0.65 ± 0.03	0.35 ± 0.01	0.005 ± 0.00005

Figure 6.

Unfolding kinetic studies of CcdB under different buffer conditions. Unfolding of CcdB follows single exponential kinetics. Kinetic traces at 2 μM protein concentration of (a) CcdB in 10 mM HEPES, pH 8.4 and (b) CcdB in 200 mM HEPES, pH 8.4 using both fluorimeter and nanoDSF are shown. All studies were carried out at 25 °C.

Photobleaching of samples during measurement

In order to examine if visible photobleaching of intrinsic fluorophores present in the protein samples occurs during the measurement time interval, the intensity of five different concentrations of HEWL (7.5, 15, 50, 100, and 500 μg/mL) was monitored for a period of 20 min at 100% excitation power at three different temperatures (20, 40, 60 °C) and at two different emission wavelengths (330 and 350 nm). There was no detectable photobleaching observed during the measurement time course, except for one condition of HEWL (500 μg/mL) at 60 °C (Fig. 8, Fig. S2). Further photobleaching was also monitored at 330 and 350 nm for five different concentration of HEWL (7.5, 15, 50, 100, and 500 μg/mL) and CcdB (7.5, 15, 50, 100, and 500 μg/mL) at two different excitation power (100% and 80%) for 60 and 150 min, respectively (Fig. S1).

Figure 8.

Absence of photobleaching of samples during measurement time scale. The intensity of 50 μg/mL of CcdB using two different capillaries was monitored for a period of 700 s at 100% excitation power at 20 °C (c) to detect any visible photobleaching of intrinsic fluorophores present in the protein samples, during the time course of measurement. The unfolding (a) and refolding (b) of the same sample is also plotted in terms of ratio of fluorescence at 350/330 nm in the same time window.

Discussion

The stability of a protein is governed by various factors. The folding process is both thermodynamically and kinetically controlled.1, 2, 3, 4 Thermodynamic stability is quantitated in terms of the standard state free energy difference between the folded and unfolded states 3. Kinetic stability on the other hand is related to the activation free energy barrier separating the folded and nonnative states.3 Fluorescence spectroscopy employing intrinsic protein fluorescence is a widely used tool to determine both thermodynamic and kinetic stability parameters.19, 20, 21 However, fluorescence measurements in conventional fluorimeters require relatively large sample volumes of about 300–800 μL. This can be a substantial limitation for studies that aim to characterize large numbers of mutants or for proteins that are difficult to express and purify. nanoDSF enables fluorescence intensity measurements in relatively small sample volumes of 15 μL. Earlier used for studying only relative thermal stabilities, the instrument has now added new software that can be used to measure both thermodynamic (PR.ChemControl) and kinetic stability (PR.TimeControl), using low concentrations (15 μg/mL) of proteins. Further, the capillary‐based measurements allow screening of 48 capillaries in parallel. This makes it possible to perform chemical denaturation experiments for two independent samples, exploring 24 different denaturant concentrations for each sample. In the present work, comparative isothermal denaturation studies have been carried out with CcdB and HEWL using both fluorimeter and nanoDSF. Additionally, isothermal refolding and unfolding kinetics for CcdB were also measured and compared with results from previous manual mixing experiments. The data indicate that nanoDSF is a usable alternative to conventional fluorimetry for measuring protein folding thermodynamics and kinetics with approximately 50‐fold lower sample requirements and over a 20‐fold decrease in measurement time/protein. Previously, a capillary‐based chemical denaturation carried out by Microscale Thermophoresis (MST)22 has been reported, but for kinetic experiments no such study has been done so far. Further in the above study, capillaries were loaded into the instrument as three sets of 16 point titrations to yield a final titration with 48 denaturant concentrations, thereby increasing the time for measurement per sample. In case of nanoDSF, 48 capillaries can be measured simultaneously, thereby further reducing the time of measurement per sample. The primary drawback for such studies is capillary cost that is currently half a euro per capillary for conventional capillaries and two euros per capillary for special sensitive capillaries that are used for proteins with few of tryptophan residues. Another drawback for the kinetic studies is the dead time for manual filling and insertion of the capillaries, which is about 15 s. This makes it difficult to simultaneously measure refolding/unfolding kinetics for multiple samples. However, these are both relatively minor issues when compared to the savings in sample amount and measurement time. An alternative to the nanoDSF is use of a fluorescence plate reader. In such cases, fluorescently labeled proteins can be used for improved sensitivity. However, labeling can affect the stability of the protein or produce experimental anomalies.23, 24, 25, 26 Further use of plastic microtiter plates can lead to sample adsorption issues that may lead to incorrect data interpretations, 26 and variations in the meniscus position between wells can also affect the measurement.

Materials and Methods

Reagents

Hen egg white lysozyme (HEWL) was obtained from USB corporation (Cleveland, USA) and was used without any further purification. HEWL was dissolved in PBS, pH 7.5. Ultrapure MB grade GdnCl was purchased from USB corporation. Extrapure AR grade urea was purchased from SRL Pvt. Ltd. (Maharashtra, India).

CcdB expression

CcdB was expressed under control of the arabinose promoter P_BAD in the pBAD24 vector using the CcdB resistant Top10Gyrase strain of E. coli, and purified using affinity chromatography as described previously.27 A single colony was inoculated into LB medium (HiMedia) and grown at 37 °C overnight. Five hundred milliliters of LB medium was inoculated with 1% of the primary inoculum and grown at 37 °C until the OD₆₀₀ reached 0.6. Cells were then induced with 0.2% (w/v) arabinose and grown at 37 °C for 5 h. Cells were harvested by centrifugation (1800g, 20 min, 4 °C). The pellet was resuspended in HEG resuspension buffer pH 7.4 (10 mM HEPES, 50 mM EDTA, 10% glycerol containing 10 mM PMSF) and sonicated, followed by centrifugation at 25,000 g, 30 min, 4 °C. The supernatant was incubated with Affi‐Gel15 (Biorad) coupled to CcdA peptide (residues 46–72) and incubated overnight at 4 °C as described previously.27 The unbound fraction was removed and washed with five times the bed volume of coupling buffer pH 8.3 (0.05 M sodium bicarbonate, 0.5 M sodium chloride). Bound CcdB was eluted with 0.2 M Glycine, pH 2.5 into a tube containing an equal volume of 400 mM HEPES, pH 8.5, 4 °C. The eluted fractions were subjected to 15% Tricine SDS‐PAGE and the protein concentration was determined using an ε₂₈₀ of 16,500 M⁻¹.cm^‐1 and 39000 M⁻¹.cm^‐1 for CcdB and HEWL, respectively.12, 28 Fractions containing pure protein were pooled to a final volume of 2 mL and were dialyzed overnight three times against 6 L of 10 mM HEPES, pH 8.4 and 200 mM HEPES, pH 8.4 using Tube‐O‐Dialyser (4 kDa MWCO, G‐Biosciences).

nanoDSF studies

Thermal unfolding experiments of the purified CcdB and HEWL were carried out by nanoDSF [Instrument weblink: (https://nanotempertech.com/prometheus/)]. nanoDSF is an advanced differential scanning fluorimetry technology which detects small changes in the fluorescence of tryptophan upon folding or unfolding in a truly label‐free manner as a function of temperature10, 11 or denaturant. The fluorescence is excited at 280 nm and is detected at 330 nm and 350 nm to derive F350/330 for data analysis. The bandwidth of measurement is 5 nm. The Prometheus NT.48 can accommodate 48 capillaries. The sample is filled into the capillaries by capillary force action and then placed into the instrument. The capillaries are single‐use and therefore no equilibration or cleaning is required. The capillaries were heated from 20 to 90 °C with a heating rate of 1 °C /min and the changes in the fluorescence ratio (F350/F330) was monitored to determine the apparent T_m. Thermal denaturation of CcdB in 10 or 200 mM HEPES, pH 8.4 was carried out at a fixed protein concentration of 47 μg/mL (2 μM) and HEWL in PBS, pH 7.5 was used at 15 μg/mL (1.05 μM). For isothermal denaturation and kinetic measurements, blanks were also subtracted. For each experiment, capillaries containing five different GdnCl concentration were used to calculate blank values. A linear fit to the data was used to estimate the fluorescence of buffer alone as a function of GdnCl concentration.

Isothermal denaturation

Equilibrium unfolding experiments of CcdB and HEWL18 were carried out by conventional fluorimetry using a JASCO FP‐6300 spectrofluorometer and nanoDSF. The changes in either the fluorescence (in case of the fluorimeter) or the fluorescence ratio (F350/F330, in case of nanoDSF using PR.ChemControl software, Prometheus NT.48) were monitored, after overnight incubation at 25 °C in different buffer conditions (10 or 200 mM HEPES, pH 8.4 for CcdB and PBS, pH 7.5 for HEWL) containing various concentrations of the denaturant, guanidium chloride (GdnCl), to determine the stability parameters for chemical denaturation (C_m). GdnCl concentrations were calculated from measurements of the refractive index using a refractometer. The data was analyzed using SigmaPlot™ version 12.5 for Windows™ scientific graphing software, from Systat Software, Inc., San Jose California USA, (www.systatsoftware.com), and the plots were fitted to a two‐state unfolding model (N↔D for HEWL and N₂↔2D for CcdB). Isothermal denaturation of CcdB in 10 or 200 mM HEPES, pH 8.4 was carried out at a fixed protein concentration of 47 μg/mL (2 μM) and HEWL in PBS, pH 7.5 was used at 15 μg/mL (1.05 μM). CcdB is a homodimer29 and HEWL is a monomer in the native state. The fraction unfolded for both CcdB and HEWL that is calculated in a similar way to the earlier studies7 is given below.

The spectroscopic signal (Y) of a protein solution (in our case, fluorescence intensity at 340 nm by fluorimeter or F₃₅₀/F₃₃₀ ratio by nanoDSF) is related to the fraction of unfolded protein (f _u) by:

$$f_{\mathrm{u}}=\left(Y-Y_{\mathrm{f}}\right)/\left(Y_{\mathrm{u}}-Y_{\mathrm{f}}\right)$$ (1)

where Y _f and Y _u are the values of Y for the folded and unfolded protein, respectively. These change linearly with the denaturant concentration ([D]), as follows:

$$Y_{\mathrm{f}}=y_{\mathrm{f}}+d_{\mathrm{f}}\left[\mathrm{D}\right]$$ (2)

$$Y_{\mathrm{u}}=y_{\mathrm{u}}+d_{\mathrm{u}}\left[\mathrm{D}\right]$$ (3)

where y _f and y _u are the folded and unfolded parameters at zero denaturant concentration. d _f and d _u are the denaturation dependence of Y for the folded and unfolded state, respectively.

Refolding and unfolding kinetics

Refolding and unfolding kinetics for CcdB were monitored by both fluorimeter8 (fluorescence intensity at 340 nm) and nanoDSF (F₃₅₀/F₃₃₀ using PR. TimeControl software, Prometheus NT.48) at 25 °C. To measure the rates of refolding, 4 μM of the dialyzed protein in either 10 or 200 mM HEPES, pH 8.4 was denatured in 3 M GdnCl and subsequently diluted to a final concentration of 1.5 M GdnCl. The changes in signal were monitored as a function of time. To measure the unfolding kinetics, protein in native buffer (10 or 200 mM HEPES, pH 8.4) was diluted into the same buffer containing 6 M GdnCl to a final concentration of 3 M GdnCl and the changes in either the fluorescence intensity at 340 nm (in case of the fluorimeter) or the fluorescence ratio (F350/F330, in case of nanoDSF) were monitored as a function of time. Refolding kinetic traces of fluorescence intensity in 1.5 M GdnCl as a function of time for CcdB in 10 mM or 200 mM HEPES, pH 8.4 were normalized from 0 to 1 between native and denatured baseline at 1.5 M GdnCl. Unfolding kinetic traces of fluorescence intensity in 3 M GdnCl as a function of time for CcdB in 10 mM or 200 mM HEPES, pH 8.4 were normalized from 0 to 1 between native and denatured baseline at 3 M GdnCl. The data was analyzed using SigmaPlot™ version 12.5 for Windows™ scientific graphing software, from Systat Software, Inc., San Jose, CA, USA, (www.systatsoftware.com), and plots were fitted to a 5 parameter equation for exponential decay for refolding (y = y₀ + a*exp(−bx) + c*exp(−dx)), yielding slow and fast phase rate constants and a 3 parameter exponential rise for unfolding (y = y₀ + a*exp[b*x]) as described previously, where x is the time (in seconds) of refolding/unfolding.8, 27

Conflict of Interest

The authors claim no conflict of interest.

Supporting information

Supplementary Figure S1 Detection of photobleaching of samples during measurement time scale using two different laser powers. The intensity of five different concentration of HEWL (7.5, 15, 50, 100 and 500 μg/mL, increasing concentration from bottom to top, A‐B and E‐F) and CcdB (7.5, 15, 50, 100 and 500 μg/mL, increasing concentration from bottom to top, C‐D and G‐H) using two different capillaries for each set, was monitored at two different laser powers (100% and 80%) for 60 and 150 minutes respectively at two different emission wavelengths (330 and 350 nm) to detect any visible photobleaching of intrinsic fluorophores present in the protein samples, during the time course of measurement.

Supplementary Figure S2. Detection of photobleaching of samples during measurement time scale at three different temperature. The intensity of five different concentration of HEWL (7.5, 15, 50, 100 and 500 μg/mL, increasing concentration from bottom to top) using two different capillaries for each set, at 100% laser power was monitored at three different temperatures (20 (A, D), 40 (B, E) and 60 (C, F)  °C) for 20 minutes at two different emission wavelengths (330 and 350 nm) to detect any visible photobleaching of intrinsic fluorophores present in the protein samples, during the time course of measurement.