Negatively Charged Submicron Heterogeneities in Aqueous Solutions of Biomolecules as Alkaline Membraneless Organelles

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The work entitled “Charged Submicron Heterogeneities in Aqueous Solutions of 2

Biomolecules as a Factor of Cell Biology”

Manuscript no: ijms-4386813

The manuscript needs the following changes before publication.

 

- Paper title should be changed to be more concise, descriptive, and keyword-rich, focusing on the main findings.

- Keywords to be changed and should be with more citable words.

- Please check grammar, word spacing and spelling mistakes.

- Introduction part should mention in details the idea behind studying SMH.

- Why authors have chosen the the ζ-potential was considered equal to 0 when the measured value fell into the range from -2 to +2 mV (reference required).

- It is advisable that authors add Optical Photothermal Infrared (O-PTIR), Raman Spectroscopy or Scanning Transmission X-Ray Microscopy (STXM) for further Submicron Heterogeneities characterization

- Please discuss the values of PI for both  both free glutamic acid and arginine and in SMH

- Microfluidic Resistive Pulse Sensing (MRPS / TRPS): can be used to accurately counts and sizes submicron heterogeneities on a particle-by-particle basis

- Please revise references to match journal style and use more recent references (you have references since 1972)- please check ref 3 vs ref 4.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

The manuscript needs the following changes before publication.

 - Paper title should be changed to be more concise, descriptive, and keyword-rich, focusing on the main findings.

The authors' response:

We have changed the title to the following:

Negatively Charged Submicron Heterogeneities in Aqueous Solutions of Biomolecules as Alkaline Membraneless Organelles

Keywords to be changed and should be with more citable words.

The authors' response:

We have changed «zeta-potential» to «particle charge»; water to «structure of water solution»; «biomolecules» to «biomolecular interactions»

- Please check grammar, word spacing and spelling mistakes.

The authors' response:

The text has been revised by a professional translator.

- Introduction part should mention in details the idea behind studying SMH.

The authors' response:

We tried to express our idea in the last three paragraphs of the Introduction. After this remark, the last paragraph of the Introduction was highlighted and supplemented.

- Why authors have chosen the the ζ-potential was considered equal to 0 when the measured value fell into the range from -2 to +2 mV (reference required).

The authors' response:

The standard method for determining the zeta-potential of charged particles is based on the registration of their motion by optical means. At zero speed (zero zeta-potential), this method does not work. Therefore, we had to take a certain interval near 0, when hitting which we considered the zeta potential to be 0, strictly speaking, close to 0. Since this was not a one-time measurement, as a result of averaging we obtained a statistically justified pH value at which the isoelectric point is reached.

- It is advisable that authors add Optical Photothermal Infrared (O-PTIR), Raman Spectroscopy or Scanning Transmission X-Ray Microscopy (STXM) for further Submicron Heterogeneities characterization

The authors' response:

We are grateful to the distinguished reviewer for suggestions on further study of the SMH. It would provide additional information. In this paper, when planning the study, we relied on the experience of previous researchers, according to which light scattering methods are the most informative. In particular, classical methods such as small-angle X-ray scattering [12] and small-angle neutron scattering [13] proved unsuitable for the study of SMH.

- Please discuss the values of PI for both  both free glutamic acid and arginine and in SMH

The authors' response:

We've updated the text as follows: Apparently, the presence of SMH pI in the acidic region is typical for all amino acids, including basic (arginine) and acidic (glutamic acid) amino acids. This indicates the common nature of the formed SMH in aqueous solutions of amino acids.

- Microfluidic Resistive Pulse Sensing (MRPS / TRPS): can be used to accurately counts and sizes submicron heterogeneities on a particle-by-particle basis

The authors' response:

In our experience, microfluidic conditions themselves affect the characteristics of SMH [19].

- Please revise references to match journal style and use more recent references (you have references since 1972)- please check ref 3 vs ref 4.

The authors' response:

We could not ignore the researchers of the effect, Vuks and Shurupova, who first experimentally discovered SMH in 1972. But apart from them, references of recent studies were used: 18,28,36,17,34,23,27,43,44,47,57

Reviewer 2 Report

Comments and Suggestions for Authors

1. The manuscript describes novel and very important observations in a relatively new and clearly underexplored area of research related to the study of structure of "true" solutions that contain "submicron heterogeneities" (SMH) (in authors' own terminology). Every new piece of evidence that clarifies the nature of this counter-intuitive solute structuring is clearly worth publishing.

 

2. The study was adequately designed, used standard methods and allowed the authors to reveal new features of the SMH present in aqueous solutions of various representative biomolecules of low molecular mass (seven amino acids, ATP, monosaccharide glucose and disaccharide sucrose). Specifically, the authors measured isoelectric points of the SMH (pI 2.4–4) and their zeta potentials, which were found to be negative similarly to other related systems reported earlier. Using the measured values of the zeta potential of SMH the authors were able to estimate the concentration of hydroxide anions inside the SMH, which corresponded to alkaline conditions featured by pH 7–10.

 

3. The main message of the manuscript is that the charged SMH in aqueous solutions of biomolecules should be considered as new players inside cells. The authors argue that since the cytosol of cells is an aqueous solution of various biomolecules and since all types of low-molecular biomolecules in water can organize into SMH, the latter must also be formed inside cells, although this has not yet become a generally accepted fact. The authors propose a hypothesis that SMH might be considered as a new class membraneless organelles. The presence of compact regions (SMH) with an alkaline pH inside the cell is a fundamentally new factor in cell biology, which undoubtedly may have important consequences.

 

4. The manuscript should be published. In reviewer’s opinion, it will have a large impact.

 

5. However, the following issues should be resolved prior to publishing:

5.1. The ref. 27 is not related to “participation of hydrophobic components” (lines 67-67). This review by Cainelli et al. should be cited when “new ways of describing chemical reactions” are mentioned (line 89).

 

5.2. A review and a more recent reference, which contain many references to related studies, should be additionally cited when “new ways of describing chemical reactions” are mentioned (line 89):

(1)       Kononov, L. O., Chemical reactivity and solution structure: on the way to a paradigm shift? RSC Adv. 2015, 5 (58), 46718-46734, https://doi.org/10.1039/c4ra17257d.

(2)       Orlova, A. V.; Malysheva, N. N.; Panova, M. V.; Podvalnyy, N. M.; Medvedev, M. G.; Kononov, L. O., Comparison of glycosyl donors: a supramer approach. Beilstein J. Org. Chem. 2024, 20, 181-192, https://doi.org/10.3762/bjoc.20.18.

 

5.3. The authors claim that “version about purely hydrophobic impurities has also been suggested [23], however, this contradicts experimental data on the hydrophilicity of SMH [19] and does not meet the requirements of their charge stability [32].” (lines 75-77).

In reviewer’s opinion, this is an absolutely incorrect statement.

Firstly, the model described in ref. 23 does suggest hydrophilicity of the external periphery of SMH (although the authors use another terminology), the hydrophobic impurities (present in trace amounts) only trigger nucleation of the solute (usually a hydrotrope); SMH are covered by solute molecules (ethanol in ref. 23) hence they are hydrophilic. A similar model has been suggested in ref. 12 and in fact corresponds to mesoscale solubilization (see ref. 22) of hydrophobic admixtures. Both models correspond well to the generally accepted model of solubilization of hydrophobic substances by hydrotropes as described in the following review:

(3)       Kunz, W.; Holmberg, K.; Zemb, T., Hydrotropes. Curr. Opin. Colloid Interface Sci. 2016, 22, 99-107, https://doi.org/10.1016/j.cocis.2016.03.005.

Note that all compounds studied in the manuscript also belong to the class of hydrotropes. The references mentioned above should also be cited in manuscript when discussing the issue of hydrophobic impurities.

Secondly, the issues of origin of charge and its stability have been discussed in detail in ref. 23. There is no contradiction at all.

The authors should discuss the results described in ref. 23 more correctly.

 

5.4. For DLS data scattering angle should be indicated. It is also highly desirable to provide the correlation functions for all solutions studied. Size distributions alone cannot prove the existence of SMH in solutions. A reader needs primary data in order to make own reasonable conclusions.

 

5.5. Line 93. ATP (studied in ref. 20) is neither a nucleic acid nor its component. Please use another word.

 

5.6. When discussing the charge (lines 122-126) and zeta potential (lines 142-143) of SMH the already mentioned ref. 23 should also be cited. Please read a detailed analysis of these issues in ref. 23 and correct the text in the manuscript accordingly.

 

5.7. Line 133. The authors should provide reasons why they attribute a fast relaxation mode, which corresponds to hydrodynamic diameter “of about 1 nm”, to “molecular fraction”. Several relevant references would be extremely helpful.

 

5.8. Lines 144 and 289. The phrases zeta-potential was “not determined” (line 144) and “not measured” (line 289) mean that there were no attempts to measure. Probably, the authors wanted to say that they could not measure the corresponding potentials due to their low values.

 

5.9. Did the authors observe coagulation or further aggregation of SMH at pH values corresponding to their zero-charge (at pH = pI)? Please indicate the hydrodynamic diameters of the light-scattering objects at these pH values and/or provide other information on light scattering of these solutions (SLS/DLS)? This phenomenon is known for other SMH. See ref. 23 and the following reference:

(4)       Rak, D.; Sedlák, M., On the mesoscale solubility in liquid solutions and mixtures. J. Phys. Chem. B 2019, 123 (6), 1365-1374, https://doi.org/10.1021/acs.jpcb.8b10638.

 

5.10. Lines 301-302. The authors claim that the “negative charge of SMH can only be provided by the presence of OH-anions.” However, the situation is not that simple. Alternative explanations have been suggested in the literature; this issue is discussed in detail in ref. 23. The authors should exercise more caution.

 

5.11. When discussing the issue of nano-bubbles the authors should cite critical reviews on this subject:

(5) Sedlák, M., Surfactant-free self-assembled mesoscale structures in multicomponent mixtures comprising solids, liquids, and gases: nanoparticles, nanodroplets, and nanobubbles. Front. Soft Matter 2023, 3, 1225709, https://doi.org/10.3389/frsfm.2023.1225709.

(6) Chen, C.; Gao, Y.; Zhang, X., The Existence and Stability Mechanism of Bulk Nanobubbles: A Review. Nanomaterials 2025, 15 (4), 314, https://doi.org/10.3390/nano15040314.

 

5.12. A header in Table 2 (line 170) claims that water with pH 7 was used. Did the authors measure the pH values of the pure water samples used and the resulting solutions? Did the authors measure the pH values of the water samples, which contained 150 mM KCl, and the resulting solutions? Did the authors attempt to exclude contact of the solutions studied with atmospheric carbon dioxide? It is well known that the pH value of ultra-pure water (Milli-Q grade) rapidly changes upon contact with air.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

1. The manuscript describes novel and very important observations in a relatively new and clearly underexplored area of research related to the study of structure of "true" solutions that contain "submicron heterogeneities" (SMH) (in authors' own terminology). Every new piece of evidence that clarifies the nature of this counter-intuitive solute structuring is clearly worth publishing.

 

1. The study was adequately designed, used standard methods and allowed the authors to reveal new features of the SMH present in aqueous solutions of various representative biomolecules of low molecular mass (seven amino acids, ATP, monosaccharide glucose and disaccharide sucrose). Specifically, the authors measured isoelectric points of the SMH (pI 2.4–4) and their zeta potentials, which were found to be negative similarly to other related systems reported earlier. Using the measured values of the zeta potential of SMH the authors were able to estimate the concentration of hydroxide anions inside the SMH, which corresponded to alkaline conditions featured by pH 7–10.

 

1. The main message of the manuscript is that the charged SMH in aqueous solutions of biomolecules should be considered as new players inside cells. The authors argue that since the cytosol of cells is an aqueous solution of various biomolecules and since all types of low-molecular biomolecules in water can organize into SMH, the latter must also be formed inside cells, although this has not yet become a generally accepted fact. The authors propose a hypothesis that SMH might be considered as a new class membraneless organelles. The presence of compact regions (SMH) with an alkaline pH inside the cell is a fundamentally new factor in cell biology, which undoubtedly may have important consequences.

 

1. The manuscript should be published. In reviewer’s opinion, it will have a large impact.

The authors' response

We are grateful to the distinguished reviewer for such a careful study of our manuscript and a deep understanding not only of the work done, but also of the significance of the results obtained for biology.

1. However, the following issues should be resolved prior to publishing:

5.1. The ref. 27 is not related to “participation of hydrophobic components” (lines 67-67). This review by Cainelli et al. should be cited when “new ways of describing chemical reactions” are mentioned (line 89).

The authors' response

Thanks for pointing out the error. The link has been moved.

5.2. A review and a more recent reference, which contain many references to related studies, should be additionally cited when “new ways of describing chemical reactions” are mentioned (line 89):

(1)       Kononov, L. O., Chemical reactivity and solution structure: on the way to a paradigm shift? RSC Adv. 2015, 5 (58), 46718-46734, https://doi.org/10.1039/c4ra17257d.

(2)       Orlova, A. V.; Malysheva, N. N.; Panova, M. V.; Podvalnyy, N. M.; Medvedev, M. G.; Kononov, L. O., Comparison of glycosyl donors: a supramer approach. Beilstein J. Org. Chem. 2024, 20, 181-192, https://doi.org/10.3762/bjoc.20.18.

The authors' response

Indeed, these references should have been added, we added them on page 3.

5.3. The authors claim that “version about purely hydrophobic impurities has also been suggested [23], however, this contradicts experimental data on the hydrophilicity of SMH [19] and does not meet the requirements of their charge stability [32].” (lines 75-77).

In reviewer’s opinion, this is an absolutely incorrect statement.

Firstly, the model described in ref. 23 does suggest hydrophilicity of the external periphery of SMH (although the authors use another terminology), the hydrophobic impurities (present in trace amounts) only trigger nucleation of the solute (usually a hydrotrope); SMH are covered by solute molecules (ethanol in ref. 23) hence they are hydrophilic. A similar model has been suggested in ref. 12 and in fact corresponds to mesoscale solubilization (see ref. 22) of hydrophobic admixtures. Both models correspond well to the generally accepted model of solubilization of hydrophobic substances by hydrotropes as described in the following review:

(3)       Kunz, W.; Holmberg, K.; Zemb, T., Hydrotropes. Curr. Opin. Colloid Interface Sci. 2016, 22, 99-107, https://doi.org/10.1016/j.cocis.2016.03.005.

Note that all compounds studied in the manuscript also belong to the class of hydrotropes. The references mentioned above should also be cited in manuscript when discussing the issue of hydrophobic impurities.

Secondly, the issues of origin of charge and its stability have been discussed in detail in ref. 23. There is no contradiction at all.

The authors should discuss the results described in ref. 23 more correctly.

The authors' response

In [23], solutions were considered in which SMH with a high zeta potential, up to -120 mV, were formed. This explains the stability of the SMH. However, the papers to which we referred give an example of other solutions with a zeta potential of less than 30 mV, which cannot provide colloidal stability. Since the same mechanism is assumed, the latter refutes the statement in [23]. In addition, the mechanism of charge appearance in the systems under consideration was not explained in [23]. For example, in [23] the authors said the following: «"... the origin of the spontaneous charging of hydrophobic interfaces remains a mystery."»

Nevertheless, our controversial statement has been deleted.

The references indicated by the reviewer have been added.

5.4. For DLS data scattering angle should be indicated. It is also highly desirable to provide the correlation functions for all solutions studied. Size distributions alone cannot prove the existence of SMH in solutions. A reader needs primary data in order to make own reasonable conclusions.

The authors' response

The details of the DLS measurement have been added to Section 3.2: A laser with a wavelength of 451 nm and a power of 50 mW was used, and light scattering was measured at an angle of 140°. Correlation functions were added to Figure 1 and Supplemental material.

5.5. Line 93. ATP (studied in ref. 20) is neither a nucleic acid nor its component. Please use another word.

The authors' response

It was changed to a nucleotide

5.6. When discussing the charge (lines 122-126) and zeta potential (lines 142-143) of SMH the already mentioned ref. 23 should also be cited. Please read a detailed analysis of these issues in ref. 23 and correct the text in the manuscript accordingly.

 The authors' response

At this point of the manuscript, experimental data on the charge characteristics of SMH in aqueous solutions of biomolecules are discussed. In [23], solutions of ethanol and linear alkanes were considered; they do not belong to biomolecules.

5.7. Line 133. The authors should provide reasons why they attribute a fast relaxation mode, which corresponds to hydrodynamic diameter “of about 1 nm”, to “molecular fraction”. Several relevant references would be extremely helpful.

 The authors' response

References have been added.

5.8. Lines 144 and 289. The phrases zeta-potential was “not determined” (line 144) and “not measured” (line 289) mean that there were no attempts to measure. Probably, the authors wanted to say that they could not measure the corresponding potentials due to their low values.

 The authors' response

We are grateful to the distinguished reviewer for this comment and have corrected the text.

5.9. Did the authors observe coagulation or further aggregation of SMH at pH values corresponding to their zero-charge (at pH = pI)? Please indicate the hydrodynamic diameters of the light-scattering objects at these pH values and/or provide other information on light scattering of these solutions (SLS/DLS)? This phenomenon is known for other SMH. See ref. 23 and the following reference:

(4)       Rak, D.; Sedlák, M., On the mesoscale solubility in liquid solutions and mixtures. J. Phys. Chem. B 2019, 123 (6), 1365-1374, https://doi.org/10.1021/acs.jpcb.8b10638.

  The authors' response

With a zero charge SMH aggregation is possible. However, the purpose of this work was to determine the isoelectric point, and the temporal dynamics of the size was not determined.

5.10. Lines 301-302. The authors claim that the “negative charge of SMH can only be provided by the presence of OH-anions.” However, the situation is not that simple. Alternative explanations have been suggested in the literature; this issue is discussed in detail in ref. 23. The authors should exercise more caution.

 The authors' response

In [23], the issue of the appearance of a negative charge on the SMH is discussed. However, the authors themselves wrote the following: "... the origin of the spontaneous charging of hydrophobic interfaces remains a mystery." One of the versions refers to the redistribution of OH-, as in our case. The second version relates to the redistribution of electron densities near the hydrophobic surface. In this case, the authors of [23] discussed the appearance of a charge on a hydrophobic surface. Which does not correspond to our case.

5.11. When discussing the issue of nano-bubbles the authors should cite critical reviews on this subject:

(5) Sedlák, M., Surfactant-free self-assembled mesoscale structures in multicomponent mixtures comprising solids, liquids, and gases: nanoparticles, nanodroplets, and nanobubbles. Front. Soft Matter 2023, 3, 1225709, https://doi.org/10.3389/frsfm.2023.1225709.

(6) Chen, C.; Gao, Y.; Zhang, X., The Existence and Stability Mechanism of Bulk Nanobubbles: A Review. Nanomaterials 2025, 15 (4), 314, https://doi.org/10.3390/nano15040314.

 The authors' response

The references suggested by the reviewer have been added.

5.12. A header in Table 2 (line 170) claims that water with pH 7 was used. Did the authors measure the pH values of the pure water samples used and the resulting solutions? Did the authors measure the pH values of the water samples, which contained 150 mM KCl, and the resulting solutions? Did the authors attempt to exclude contact of the solutions studied with atmospheric carbon dioxide? It is well known that the pH value of ultra-pure water (Milli-Q grade) rapidly changes upon contact with air.

The authors' response

We did not measure the pH of pure water, because it is well known that water at pH = 7 quickly acidifies due to the dissolution of carbon dioxide. We titrated ready-made aqueous solutions of biomolecules until they reached pH=7.

We did not protect the solutions from carbon dioxide, on the contrary, the used water stood for a day to saturate with air (this was added to the description of the Methods). Also, aqueous solutions have a certain buffer capacity and they are no longer significantly affected by the dissolution of carbon dioxide.