PONE-D-24-20499Interaction of 4-ethyl phenyl sulfate with BSA: Experimental and molecular docking studiesPLOS ONE

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Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

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Reviewer #1: The manuscript by Payal Gulati et al. entitled “Interaction of 4-ethyl phenyl sulfate with BSA: Experimental and molecular docking studies” describes the interaction of BSA with 4-EPS using different techniques.

The authors conclude that this interaction study may be helpful in devising strategies for the treatment of chronic kidney disease and other neuro related diseases wherein interaction of 4-EPS and BSA matters.

In my opinion the work should be improved. Data presented demonstrate the link between BSA and 4-EPS but it was already known news. There are no predictions of how this link might be severed. The work does not bring anything new to readers.

I admit this manuscript with major revision.

In particular:

- I would like to suggest improving the introduction. The introduction does not seem to have a linearity, the different parts do not seem connected to each other.

- The authors declare that the BSA-4-EPS conjugate was purified from unreacted material and by-products, but they don't describe how they do it (2.2 Conjugate preparation paragraph).

- I would suggest including the control of the experiments shown in Figure 1 and Figure 2. I would like to suggest demonstrating that the decrease in fluorescence intensity is due to the interaction with the molecule and not to a change in volume or other.

- In figures 1a and 2b I would like to suggest inserting the legend with the concentrations for a more immediate view.

- In figure 3, EPS is shown and not 4-EPS and is not readable. To compare the graphs, they should all start from the same percentage of transmittance.

- The resolution of the graphics is very bad

- Why were the experiments carried out at pH 4.3?

- In figure 7, the letters a and b cannot be read.

- The authors in the abstract declare that: increased level of 4-EPS in human body has also been found implicated in anxiety; in the conclusion they declare that: This technique will help in treatment of autism in patients. There is no linearity.

Reviewer #2: In the manuscript “Interaction of 4-ethyl phenyl sulfate with BSA: Experimental and molecular docking studies” the authors study the interaction of the e4-ethyl phenyl sulfate with the bovine serum albumin.

In my opinion, this work should be of interest to the readers of the Journal but I recommend the publication of this work after a major revision. In particular:

1) The title of the article contains the abbreviation BSA and not its full name. Please, the authors modify it.

2) In the abstract, is not clear the main aim of the article.

3) The introduction section is too long and it is difficult to easily understand the aim of the work. The authors declare that the selected compound is associated with autism disorder so, why the authors study this interaction with BSA and not with HSA? Please, the authors clarify it.

4) The sentence in paragraph 2.2 “confirmation of the successful…studies” should be removed because is not needed in the MM section.

5) In the MM the authors include paragraph 2.2 defined “conjugate preparation” in which they described the sample preparation (BSA + 4-ethyl phenyl sulfate) before the spectroscopy characterization. The authors describe the incubation of the BSA with different analyte concentrations. The protocol reported is not clear. Please, the authors clarify it.

Why the authors perform the incubation with an increased concentration of the analytes at pH 4.3? Have the authors studied the effect of this pH on the BSA structure? What is the effect on the binding? Have the authors information on the binding at neutral pH instead of low pH? Please, the authors clarify it.

6) In the fluorescence spectroscopy paragraph reported in the MM section, the authors have not reported the excitation wavelength value used in the steady-state experiments. Please, the authors provide it.

7) Have the authors verified the optical density (OD) of the BSA at the concentration of 2uM? This value should be between 0.05-0.1 OD at the excitation value to avoid the inner filter effect. Have the authors control it?

8) What are the positive and negative controls in the experiments? Please, the authors provide it.

9) Paragraph 3.3 starts with the figure legend, table 1, and then describes the FT-IR results. Please, the authors re-organize this paragraph. Similar problems there are for the paragraphs 3.4.1, 3.4.2, and 3.5. Please, the authors fix it.

10)In the material methods section the authors describe the absorption spectroscopy experiments and then the fluorescence spectroscopy experiments. Why in the “results and discussion” section the authors show before the florescence results and then the absorption results? This order does not have sense. Please, the authors modify it.

10) In the “results and discussion” section the authors discuss the fluorescence experiments and report the excitation value used (280 nm). At this wavelength are excite both the tryptophan and tyrosine residues present in BSA and not only the tryptophan as discussed by the authors. Normally, the binding interaction of ligands and protein is followed as a variation of the tryptophan microenvironment by excitation at 295 nm. So, the results obtained in this condition (excitation at 280nm) need to be discussed considering both fluorescence contributions (tryptophan and tyrosine). Please the authors repeat the experiments by excite the BSA at 295 nm and compare the obtained results with the data reported in the manuscript.

11) Why the fluoresce emission was recorded in the range of 300-600 nm? The second pick present in the spectra, that appears at 560 nm (double of 280nm), is the harmonic pick of the excitation (see Figure 1A). This has no sense. Please the author modifies Figure 1.

12) In Figures 2 A and 2B the wavelength range is not the same. Please, the authors fix it.

12)From the quenching experiments the authors have calculated the thermodynamic parameters of the binding between BSA and the 4-ethyl phenyl sulfate. Please, to allow the readers to better understand these results include the obtained results in a table and discuss it with the type of molecular interaction identified.

13) All the mathematical equations (Stern-Volmer, thermodynamic equations, etc.) used and reported in the “results and discussion” section should be removed and included only in the “materials and method” section. Please the authors provide it.

14) All the figure's legends should be increased. Please, the authors provide it.

15) Table 2 appears in a different layout respect to table 1. Please, the authors re-organize it.

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Reviewer #1: No

Reviewer #2: No

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Interaction of 4-ethyl phenyl sulfate with bovine serum albumin: Experimental and molecular docking studies

PONE-D-24-20499R1

Dear Dr. Payal Gulati,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Sabato D'Auria

Academic Editor

PLOS ONE

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