Protocol for evaluating drug-protein interactions based on fluorescence spectroscopy

Summary

Investigating the interactions between ligands, such as drugs, and proteins is essential to understand their functions and effects. This fluorescence spectroscopy protocol details how to analyze the effects of drugs on protein structure, including steps for solution preparation, fluorescence spectra collection, and results analysis. It is applicable to studying drug-protein interactions, binding affinity, and thermodynamic parameters as well as performing further studies on drug design.

For complete details on the use and execution of this protocol, please refer to Jalali et al.¹ and Sargolzaei et al.²

Graphical abstract

Highlights

• •Instructions for examining drug-protein interaction with fluorescence spectroscopy
• •Guidance on analyzing binding sites on a protein for a specific drug
• •Steps for calculating thermodynamic parameters in drug-protein interaction

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Investigating the interactions between ligands, such as drugs, and proteins is essential to understand their functions and effects. This fluorescence spectroscopy protocol details how to analyze the effects of drugs on protein structure, including steps for solution preparation, fluorescence spectra collection, and results analysis. It is applicable to studying drug-protein interactions, binding affinity, and thermodynamic parameters as well as performing further studies on drug design.

Before you begin

Fluorescence is a useful technique for investigating chemical environments and intermolecular interactions that can be used to understand drug-protein interactions. Fluorescence emission spectra and lifetimes are very sensitive to molecular interactions and environmental conditions.³ The following protocol describes the steps to investigate the interaction between drugs and proteins, specifically human serum albumin (HSA), using a fluorescence technique to obtain information about the binding affinity of a specific drug to HSA binding sites and the interaction between them. HSA contains the aromatic amino acids tryptophan, tyrosine, and phenylalanine, which emit fluorescence; their emission wavelengths are listed in Table 1. In proteins that contain tryptophan and other fluorescent amino acids (phenylalanine and tyrosine), the energy absorbed by phenylalanine and tyrosine is usually transferred to tryptophan, which emits fluorescence at approximately 350 nm.^4,5 It should be noted that in proteins that do not contain fluorescent amino acids, fluorescent probes can be used to measure fluorescence and investigate structural changes.⁶ In addition, fluorescence spectroscopy probes not only excitation and emission spectra but also quenching phenomena, binding propensity, and thermodynamic parameters.

Table 1.

The maximum wavelengths of fluorescence excitation and emission of aromatic amino acids

Sample	Excitation wavelength (nm, ±1 nm)	Emission wavelength (nm, ±5 nm)	Reference
Tryptophan	280	350	Pan5 and Smith et al.7
	266		Seaver et al.8
Tyrosine	275	300	Pan5 and Smith et al.7
	266		Seaver et al.8
Phenylalanine	260	280	Smith et al.7
	266		Seaver et al.8
	255		Pan5

This protocol covered sample preparation procedures, spectrum measurements, data analysis, and all critical items to be followed during the experiment. Our protocol can help researchers who intend to investigate the interaction of a drug with a protein using the fluorescence technique to perform all the steps of the experiment correctly and with the highest precision and caution.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Biological samples
Human serum albumin	Sigma-Aldrich	CAS No. 70024-90-7
Chemicals, peptides, and recombinant proteins
NaH2PO4	Sigma-Aldrich	CAS No. 13472-35-0
Na2HPO4	Sigma-Aldrich	CAS No. 10028-24-7
NaCl	Sigma-Aldrich	CAS No. 7647-14-5
KCl	Sigma-Aldrich	CAS No. 7447-40-7
HCl	Sigma-Aldrich	CAS No. 7647-01-0
NaOH	Sigma-Aldrich	CAS No. 1310-73-2
CH3CH2OH (ethanol)	Sigma-Aldrich	CAS No. 64-17-5
Drug (e.g., sertraline and buspirone)	N/A	N/A
Software and algorithms
Cary Eclipse WinFLR	Agilent Technologies	http://crosslab.chem.agilent.com/
ChemDraw Professional 18.0	PerkinElmer	http://www.perkinelmer.com/
Other
Cary Eclipse fluorescence spectrometer	Agilent Technologies	http://crosslab.chem.agilent.com/
Bain-marie	Trustrade	https://trustrade.ae/
pH meter	Jenway	https://www.coleparmer.com/
Cuvette	Hellma 108F-QS	ARTICLE NO. 108-F-10-40

Step-by-step method details

Stock solution preparation

Timing: 2–4 h

Stock solutions of drug, protein, and buffer are prepared to prepare sample solutions.

• 1.Prepare phosphate-buffered saline solution (PBS) containing NaH₂PO₄ and Na₂HPO₄.

Note: The type of buffer is selected based on the type of drug. PBS buffer can contain NaH₂PO₄, Na₂HPO₄, KCl, and NaCl, but for sertraline and buspirone, the buffer containing NaH₂PO₄ and Na₂HPO₄ was more suitable.^1,2 In general, a suitable buffer is one in which the solubility of the drug is high and ions are not formed.

Note: The pH of the buffer should be adjusted to 7.4 due to the simulation of the laboratory environment with the human body, using NaOH or HCl and measuring it with a pH meter.

• 2.Prepare protein stock solution: containing the protein and double-distilled water.
• 3.Prepare drug stock solution: containing the drug and double-distilled water.

Note: Use drugs and proteins with purity >95%.

Calculate the amounts of drug, protein, and salt using Equation 1^9,10:

$$\mathrm{m}\left(\mathrm{g}\right)=\text{MW}\times \mathrm{V}\times \mathrm{C}$$ (Equation 1)

where C is the molar concentration in mol/L or M, m is the mass of the solute in grams (g), V is the volume of the solution in liters (L), and MW is the molecular weight of the solute in g/mol.

Note: Drug stock solutions can be stored for two weeks and protein stock solution for 4–5 days at 4°C.

Sample preparation

Timing: 1–2h

Drug and protein samples are prepared for fluorescence spectroscopy.

• 4.Prepare the desired concentrations for the study using drug and protein stocks and dilution with buffer.

Note: Dilute according to Equation 2¹¹:

$${\mathrm{M}}_1{\mathrm{V}}_1={\mathrm{M}}_2{\mathrm{V}}_2$$ (Equation 2)

Where M₁ and V₁ are the concentration of the stock solution and its volume in liters, respectively; and M₂ and V₂ are the concentration and volume of the sample to be tested, respectively.

Equipment preparation

Timing: <1 h

The fluorescence spectrometer is placed in standby mode to collect spectra of drug, protein, and drug-protein solutions.

• 5.Turn on the fluorescence spectrophotometer.
• 6.Turn on the computer and run the fluorescence data acquisition software.
• 7.Set the spectral range to 100–600 nm.

Note: The range of 100–600 nm covers the emissions related to aromatic amino acids (Trp, Tyr, Phe).

• 8.Set the slit to 5/5 for HSA.
• 9.Adjusting the excitation wavelength, which is usually 280 to 295 for HSA.

Note: The emission wavelength is selected according to the protein and drug being tested.

• 10.Specify the data storage paths for each test group.
• 11.Preparing and adjusting the temperature of the bain-marie before adding protein to the samples.

Collecting fluorescence spectra

Timing: 2–3 h

Fluorescence spectra are collected to obtain information about the drug-protein interaction.

• 12.Fill the cuvette with the buffer solution and collect the spectrum.
• 13.Remove the buffer solution from the cuvette and wash the cuvette.
• 14.Fill the cuvette with the drug solution and collect the spectrum.
• 15.Remove the solution from the cuvette and wash it.
• 16.Fill the cuvette with the protein solution and collect the spectrum.
• 17.Wash the cuvette.
• 18.Add the desired amount of protein to each sample and incubate for 1 h at different temperatures in the dark in the bain-marie.

Note: Protein concentration range is determined based on the number of fluorescent amino acids. A concentration range of 1–10 μM is considered for HSA because of the presence of a tryptophan residue (Trp-214), which typically provides a strong fluorescence signal without internal filter effects or significant aggregation.

Note: The selected temperature range is usually between 25°C and 40°C, which covers the temperature of the laboratory environment during the preparation of the solution to the temperature of the human body during fever.

CRITICAL: To get more accurate results, calculate the fluorescence emission of each concentration of drug and protein for more than four temperatures.

• 19.Collect the spectrum of protein alone and drug-protein solutions at different temperatures (25°C–40°C) in separate steps.

CRITICAL: Incubating the protein alone and collecting its spectrum is critical because temperature can affect the fluorescence spectrum of proteins.

• 20.Save the spectra in the desired file.

CRITICAL: Repeat the above steps at least three times on different days and average the obtained numbers to ensure the repeatability of the test and reduce the error.

Data analysis

Timing:>1 day

Binding parameters are calculated and analyzed using equations to evaluate the drug-protein interactions.

• 21.Subtract the reference spectrum (drug solution) from the drug-protein spectrum.

Note: The subtraction of the reference spectrum from the spectrum of the drug and protein solution is done to remove the drug emission.

• 22.Select the desired regions (200–500 nm).

Note: The target areas in this study include aromatic amino acids because the highest amount of emission is related to them. Therefore, we consider the spectral region of 200–500 nm.

• 23.Analyze the obtained spectra in Excel software.
• 24.Select the maximum amount of fluorescence emission of different drug-protein concentrations.

CRITICAL: Select a certain wavelength for all concentrations.

Note: The fluorescence emission peak for HSA is usually around 350 nm.⁴

• 25.Drawing the Stern-Volmer graph F₀ versus F and calculating the quenching constant and quench type (dynamic/static) using Equation 3⁴:

$${\mathrm{F}}_0/\mathrm{F}=1+{\mathrm{K}}_{\text{sv}}\left[\mathrm{Q}\right]=1+{\mathrm{K}}_{\mathrm{q}}{\tau }_0\left[\mathrm{Q}\right]$$ (Equation 3)

where F₀ and F represent the fluorescence intensity of HSA at λ_em before and after drug addition, respectively, and [Q] is the quencher concentration. K_q is the bimolecular quenching constant and τ₀ is the fluorescence lifetime of biomacromolecules without quencher.

Note: In this experiment, drug molecules are the only quencher present in the solution.

• 26.Plot the log (F₀-F)/F versus the log[Q] to obtain the binding affinity of the drug and protein. The binding constant (K_b) and number of binding sites (n) can be obtained using Equation 4¹²:

$$\log \left({\mathrm{F}}_0-\mathrm{F}\right)/\mathrm{F}=\log {\mathrm{K}}_{\mathrm{b}}+\text{nlog}\left[\mathrm{Q}\right]$$ (Equation 4)

• 27.Drawing the Van’t Hoff plot and calculating the thermodynamic parameters related to drug-protein binding (ΔH°, ΔS°, and ΔG°) using the Van’t Hoff plot and its equation. Using the equation obtained from the plot and Equations 5 and 6,^13,14 these parameters can be calculated, and information about the type of connection can be obtained.

$${\text{lnK}}_{\mathrm{b}}=-\Delta {\mathrm{H}}^{\mathrm{o}}/\text{RT}+\Delta {\mathrm{S}}^{\mathrm{o}}/\mathrm{R}$$ (Equation 5)

where ΔH° is enthalpy, ΔS° is entropy, R is the gas constant, and T is temperature.

The equation obtained from the Van’t Hoff plot is equivalent to Equation 5. The slope of graph is equal to ΔH°/RT and the y-intercept of the line is equal to ΔS°/R. Therefore, ΔH° and ΔS° can be calculated.

• 28.Using the data obtained from Equation 5 in step 27 (ΔH° and ΔS°) and Equation 6, ΔG° (Gibbs free energy change) can be calculated.

$$\Delta {\mathrm{G}}^{\mathrm{o}}=\Delta {\mathrm{H}}^{\mathrm{o}}-\mathrm{T}\Delta {\mathrm{S}}^{\mathrm{o}}=-\text{RTlnK}$$ (Equation 6)

Expected outcomes

By following this protocol, the binding constant (K_b), the number of binding sites of the target drug on the protein (n) and the thermodynamic parameters in drug- protein interactions can be obtained.

In this experiment, it is expected that the fluorescence emission plot of the drug and protein will be as shown in Figure 1 and that the emission rate will decrease with an increase in drug (quencher) concentration. The Stern-Volmer plot (Figure 2) shows the type of protein quenching (dynamic or static) due to the interaction with the tested drug, and the slope of the plot shows the Stern-Volmer constant (K_sv). If the slope of the graph increases with increasing temperature, it indicates dynamic quenching; if the slope of the graph decreases with increasing temperature, it indicates static quenching. In addition, the slope of the log(F₀-F)/F graph (Figure 3) shows the number of binding sites (n) for the drug on the protein, where the line crosses the y-axis, indicating the binding affinity of the drug to the protein (K_b). Moreover, the thermodynamic parameters can be calculated using the Van’t Hoff equation (Figure 4). Examples of graphs obtained from our recent studies based on drug-protein (sertraline-HSA and buspirone-HSA) fluorescence emission analysis are provided below.^1,2

Figure 1.

Fluorescence spectra of HSA with anxiolytic drugs

Fluorescence emission spectra of HSA upon interaction with (A) sertraline and (B) buspirone, illustrating changes in fluorescence intensity related to drug binding interactions.

Figure 2.

The Stern-Volmer plots for analyzing the fluorescence quenching of HSA

Stern-Volmer plots used to determine the quenching mechanism of HSA upon interaction with (A) sertraline and (B) buspirone at four different temperatures.

Figure 3.

Binding analysis of sertraline and buspirone to HSA

Plot of log (F₀-F)/F versus log[Q] illustrating the determination of the binding constant (K_b) and the number of binding sites for (A) sertraline and (B) buspirone on HSA at four different temperatures.

Figure 4.

The Van’t Hoff plot for analyzing thermodynamic parameters

Van’t Hoff plot illustrating how temperature affects the binding interactions and thermodynamic parameters of HSA with (A) sertraline and (B) buspirone.

Limitations

Since this protocol is used for the interaction of drugs and proteins in aqueous solutions, we have limitations in implementing this protocol for proteins that are insoluble at high concentrations or proteins in different buffers with high salt concentrations. In addition, this protocol considers the effects of temperature changes on fluorescence emission and drug-protein interactions, whereas other environmental effects that could have an effect cannot be measured with it. Also, this protocol examines the changes and effects of drugs on proteins within a limited period of time.

Troubleshooting

Problem 1

The fluorescence signal is weak or absent at lower temperatures (section “collecting fluorescence spectra”, step 19).

Potential solution

• •Check the fluorophore and protein concentrations, excitation light intensity, compound purity, and temperature, and provide optimal conditions.
• •Clean the cell completely and repeat the test.

Problem 2

There are unexpected peak wavelengths in the fluorescence spectrum temperatures (section “collecting fluorescence spectra”, step 19).

Potential solution

• •Check the purity of the solution.
• •Check the type of buffer, its pH, and the absence of ions and, if necessary, change the buffer or its pH.
• •Repeat the test under optimal and controlled conditions.

Problem 3

Fluorescence intensity is either too high or too low (section “collecting fluorescence spectra”, step 19).

Potential solution

• •Check the fluorophore concentration, excitation light intensity, compound purity, and temperature and provide optimal conditions.
• •Change the device slit according to the fluorescence intensity of the desired protein.

Problem 4

The fluorescence emission peak at a given concentration is different in each experiment repetition (section “collecting fluorescence spectra”, step 20).

Potential solution

• •Increasing accuracy in the preparation of solutions and ensuring the correct calculation of the concentration of each solution.
• •Check environmental conditions such as temperature and that the device is not exposed to sunlight or nearby heat sources.
• •Repeat the test.

Problem 5

The delay in fluorescence measurement causes the temperature of the solution to change (section “collecting fluorescence spectra”, step 18).

Potential solution

• •Place the bain-marie next to the fluorescence device so that the transfer of the solution does not significantly affect the temperature of the solution.
• •Repeat the test.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Javad Sargolzaei (j-sargolzaei@araku.ac.ir).

Technical contact

Further information and requests about the technical specifics of performing the protocol should be directed to and will be fulfilled by the technical contact, Elaheh Jalali (Elaheh.jalali75@gmail.com).

Materials availability

This protocol does not generate new reagents.

Data and code availability

This protocol does not generate any unique datasets or code.

Acknowledgments

We would like to thank the authorities at Arak University for their financial support, which made this research possible.

Author contributions

Conceptualization, E.J.; methodology, E.J. and J.S.; investigation, E.J.; visualization, E.J.; writing – original draft, E.J.; writing – review and editing, J.S. and E.J.; supervision, J.S.

Declaration of interests

The authors declare no competing interests.