Electroenzymatic Model System for the Determination of Catalytic Activity of Erwinia carotovora L-Asparaginase

Round 1

Reviewer 1 Report

 

Reviewing the second version of the manuscript by Victoria Shumyantseva et al., I find it suitable for publication.

The paper describes some preliminary results; however, it may be considered a proof of concept. For a full report, the authors should examine a much more comprehensive range of amino acids, carefully analyze the outcomes, check reproducibility and compare their electrochemical method for determining the catalytic activity of L-asparaginase with that elucidated by other means.

Anyhow, the currently presented results convinced me that the proposed system could be developed into an applicable analytical procedure in the future.

Author Response

Manuscript ID: processes-1797584

 

Dear Dr. Song!

We thank you and reviewer 1, 2 and 3 for the careful and detailed analysis of our paper “Electroenzymatic model system for the determination of catalytic activity of Erwinia carotovora L-asparaginase” and understanding the novelty of our research. It was hard work, however proposed electrochemical approach is suitable for the screening of L-asparaginase possessing catalytic activity.

 

Rev1

Reviewing the second version of the manuscript by Victoria Shumyantseva et al., I find it suitable for publication. The paper describes some preliminary results; however, it may be considered a proof of concept. For a full report, the authors should examine a much more comprehensive range of amino acids, carefully analyze the outcomes, check reproducibility and compare their electrochemical method for determining the catalytic activity of L-asparaginase with that elucidated by other means. Anyhow, the currently presented results convinced me that the proposed system could be developed into an applicable analytical procedure in the future.

We thank Reviewer 1 for the analysis of our paper. We really described our preliminary results concerning the approach for the determination of enzymatic activity of asparaginase by means of electrochemistry as a proof of concept. We plan to continue this investigation as PhD theses using new composite materials for the modification of working electrodes, several types of enzymes from different sources, and a range of amino acids.

 

Reviewer 2

Comments and Suggestions for Authors Shumyantseva et al. reported a study using electroenzymatic model system for the determination of L-asparaginase activity. They found that the substrate-binding can affect the oxidation of the enzyme, thus the electro-oxidation signal can be correlated with the amount of substrate, finally generating the measurement of the Km value of the enzyme. Some flaws should be improved by additional experiments and text revision. Details are below:

1. The design of the experiment in Figure 5 has some problems. Only one point has an Asn concentration below the estimated Km. In enzymatic reaction, lower substrate concentrations will have larger slopes in this curve, therefore more experimental data are needed at low substrate concentrations.

In our paper, we have shown that our results may be useful for the determination of asparaginase activity by means of electrochemical approach and for the screening of L-asparaginase possessing catalytic activity. We plan to work further with asparaginases from different sources, mutant forms of enzyme and to use a range of amino acids. Resulting curve permits to show the influence of substrate L-asparagine on oxidation current of enzyme and evaluate the impact of L-asparagine on oxidation current of protein as measure of catalytic activity.

2. The theory behind Figure 5 and the estimated Km value is not clear. The equation used in curve fitting should be shown in the manuscript.

For the determination of Michaelis constant Km, we used low concentration of substrate in the range of 200 -2500 µM.  The determination of Km was done by fitting experimental curves to obtain this parameter using the Michaelis-Menten model. Km was calculated based on determination of this value as concentration of substrate which produce half of maximum rate (in our case, half of maximum oxidation current). We used the range of concentration as 200, 400, 800, 1600, 2000, 2400 µM.

The dependence presented in Fig. 5 was approximated to a hyperbolic function (R² = 0.968) in accordance with the Michaelis-Menten equation for steady-state enzyme kinetics from the dependences of the catalytic current as a measure of reaction rate on concentration using nonlinear regression analysis [49, 50].

49. Rusling, J.F.; Wang, B.; Yun, S. Electrochemistry of redox enzymes. In Bioelectrochemistry: Fundametals, Experimental Techniques and Applications; Bartlett, P.N., Ed.; John Wiley & Sons, Ltd.: New Jersey, NJ, USA, 2008; pp. 39–85. https://doi.org/10.1002/9780470753842.ch2.
50. Shumyantseva V.V., Bulko T.V., , Koroleva P.I., Shikh E.V., MakhovaA.A., Kisel M.S., Haidukevich I.V., Gilep A.A. Human Cytochrome P450 2C9 and its polymorphic modifications: electroanalysis, catalytic properties, and approaches to the regulation of enzymatic activity. Processes 2022, 10, 383.

We really described our preliminary results concerning the approach for the determination of enzymatic activity of asparaginase by means of electrochemistry as a proof of concept. We plan to continue this investigation as PhD theses using several types of enzymes from different sources, and a range of amino acids.

 

 

3. Line 254: Km was estimated as 400 uM by fitting. How many errors of this value?

Answer

For the determination of Km the values are the means from at least 3 experiments ± S.D. We recalculated the value of Km as 600±70 µM. 

4. Lines 53-60 (References 10-12): These cited methods are not widely accepted methods with broad applications, which are inappropriately cited in the Introduction. These references should be removed. Other methods, such as absorbance, fluorescence, and other convenient and well-established techniques should be cited in this paragraph.

Answer

We agree with Reviewer 2 that methods mentioned in reference 10-12 is not widely accepted methods with broad applications. Electroanalysis is also new approach for these purposes.  However, new methods are developing rapidly, so we prefer to cite these methods for the understanding the role of nanotechnology in enzymology.

Physical, physical and chemical and nanotechnological approaches have also contributed to the development of detection methods for the estimation of enzyme catalytic activity. We mentioned these methods as developing new approaches for the determination of enzyme catalytic activity. We cited “classical” methods such as absorbance, fluorescence for the determination of catalytic activity on p. 1-2 of manuscript (references 1,6,7,8).  

5. The disadvantage and future direction of this method should be discussed in the manuscript.

We added in the manuscript the disadvantage and future direction of this method (p. 11 of manuscript).

 However, the electrochemical approach depends on the type of electrode, electrode modifier and accessibility of substrate to active center of enzyme. Future direction of this method is the construction of new composite materials for the modification of working electrodes with enhanced sensitivity to the electrochemical oxidation of amino acids, investigation of mutant forms of asparaginase from different sources, a range of amino acids as substrates, and inhibitors of this enzyme.

 

6. Line 296: WhyTyr29 is the most important candidate? Is it close to the active site/substrate-binding site? A structure figure illustrating the active site and potential oxidized residues may be helpful.

Tyr29 is a crucial amino acid in the active cite of bacterial L-asparaginases. The recent publication of our colleagues (reference 33. Pokrovskaya, M.V.; Pokrovsky, V.S.; Aleksandrova, S.S.; Sokolov, N.N.; Zhdanov, D.D. Molecular Analysis of L-Asparaginases for Clarification of the Mechanism of Action and Optimization of Pharmacological Functions. Pharmaceutics 2022, 14, 599. https://doi.org/10.3390/pharmaceutics14030599) describes in details the active sites and molecular organization of mechanism of action of L-asparaginases. For this reason, we decided not to repeat the figures of this paper. Tyr25 corresponds to Tyr29 of L-asparaginase from Erwinia carotovora and involved in substrate binding and release of reaction product [38].  We suppose that L-asparagine can change the electrochemical signature at the time of location at the active center on the enzyme by interaction with Tyr29.

7. Line 14, The minus in “0.5-0.6” is incorrectly displayed in the PDF file.

The oxidation peak corresponded to  0.5÷0.6 V. 

8. Lines 92-93: The meaning of “the ability and sensitivity of amino acids of the polypeptide backbone to conformational changes” is unclear. What is the meaning of “the amino acids of the polypeptide backbone”?

We corrected this sentence as

“In our investigation, the ability and susceptibility of the protein to conformational changes registered by electrochemical oxidation were used to estimate the catalytic activity of ASNase.” (p. 4 of manuscript).

 

9. Lines 298-299: The last sentence is incomplete.

We corrected this sentence. Thank you for this comment.

10. Lines 286-291: This paragraph is the background information of ASNase, so this part should be moved into the Introduction or deleted.  

In accordance with Reviewer 2 recommendation, we moved this paragraph into introduction (p. 2 of manuscript).

 

 

 

Rev3

Shumyantseva et al. report on the development of a method to measure the activity of the Erwininia L-asparaginase (ASNase) based on the electrooxidation of amino acids from the backbone of the ASNase. This electrochemical behavior was obtained by assaying the immobilized enzyme on modified single wall carbon nanotubes as the sensing element. The authors demonstrated that the  oxidation current of the ASNase was reduced in the presence of asparagine in a concentration dependent manner but was unaffected by the presence of the non-substrate glycine. ASNase has been utilized in medicine as a pharmacological antitumor agent and in the food industry in preventing formation of acrylamide in processed foods. Current methods used to measure ASNase activity is based on utilizing with HPLC, circular dichroism, electrophoresis assays, or colorimetric assay measuring presence of substrate asparagine with hydroxylamine or presence of product ammonia with Nessler’s reagent.    Specific Comments: (1)   

This method is effective for examining the activity ASNase for medical and food industry uses. In expanding it use for enzymatic and kinetic utilization, did the authors look at the effect of aspartate or D-asparagine on the electrooxidation properties? 

Answer

 We thank Reviewer 3 for this suggestion.  We plan to continue this investigation as PhD theses using several types of enzymes from different sources, mutant forms of asparagine and a range of amino acids, such as glutamine, aspartate and D-asparagine. In our present paper, we did not examine aspartate and D-asparagine. The main goal of this paper was to demonstrate suitability of electro analysis for the determination of enzyme activity to substrate L-asparagine.

 

(2) Are the Michaelis constants reported in this manuscript closer to a binding constant than a Km affinity constant?

Thank you for this suggestion. However, it is a complicated question. Tyr29 of L-asparaginase from Erwinia carotovora is involved in substrate binding and release of reaction product  (reference 33. Pokrovskaya, M.V.; Pokrovsky, V.S.; Aleksandrova, S.S.; Sokolov, N.N.; Zhdanov, D.D. Molecular Analysis of L-Asparaginases for Clarification of the Mechanism of Action and Optimization of Pharmacological Functions. Pharmaceutics 2022, 14, 599. https://doi.org/10.3390/pharmaceutics14030599) As we assumed that catalytic activity can be expressed as oxidation current of L-asparaginase, based on Tyr29 electrochemical oxidation, we can determined Michaelis constant. If we assumed that catalytic current is a measure of catalytic activity, we can use the term Michaelis constant Km.  

(3)   Were the kinetic properties of glutamine determined with this method? 

Answer

In our paper, we have shown that L-asparagine and L-glutamine reduce oxidation peak current of asparaginase in different manner, such as L-asparagine - 23%, for Gln - 43%, and Gly - 0%. However, we did not determined kinetic parameters of L-glutamine in this paper. The aim of this paper was to demonstrate applicability of electrochemical approach for the analysis of catalytic activity based on sensitivity of protein to different amino acids. We plan to work with this enzyme in more details, using different types of asparaginases, and a range of amino acids and substrates, such as D-asparagine, succinic acid monoamide and asparaginyl-tRNA.

4)   I assume the electrochemical method could also be used to look at the effects of competitive inhibitors to asparagine binding. Is this correct?

We thank Reviewer 3 for this suggestion. I hope that it is possible. The blocking of binding and catalytic sites of asparaginase must prevent binding with L-asparagine.  We plan to include this investigation in our future work as PhD theses.

 

 

Sincerely,

Victoria Shumyantseva, Dr Sci., Prof,

Member of Editorial Board of Processes

 

Author Response File: Author Response.docx

Reviewer 2 Report

Shumyantseva et al. reported a study using electroenzymatic model system for the determination of L-asparaginase activity. They found that the substrate-binding can affect the oxidation of the enzyme, thus the electro-oxidation signal can be correlated with the amount of substrate, finally generating the measurement of the Km value of the enzyme. Some flaws should be improved by additional experiments and text revision. Details are below:

 1.       The design of the experiment in Figure 5 has some problems. Only one point has an Asn concentration below the estimated Km. In enzymatic reaction, lower substrate concentrations will have larger slopes in this curve, therefore more experimental data are needed at low substrate concentrations.

2.       The theory behind Figure 5 and the estimated Km value is not clear. The equation used in curve fitting should be shown in the manuscript.

3.       Line 254: Km was estimated as 400 uM by fitting. How many errors of this value?

4.       Lines 53-60 (References 10-12): These cited methods are not widely accepted methods with broad applications, which are inappropriately cited in the Introduction. These references should be removed. Other methods, such as absorbance, fluorescence, and other convenient and well-established techniques should be cited in this paragraph.

5.       The disadvantage and future direction of this method should be discussed in the manuscript.

6.       Line 296: WhyTyr29 is the most important candidate? Is it close to the active site/substrate-binding site? A structure figure illustrating the active site and potential oxidized residues may be helpful.

7.       Line 14, The minus in “0.5-0.6” is incorrectly displayed in the PDF file.

8.       Lines 92-93: The meaning of “the ability and sensitivity of amino acids of the polypeptide backbone to conformational changes” is unclear. What is the meaning of “the amino acids of the polypeptide backbone”?

9.       Lines 298-299: The last sentence is incomplete.

10.   Lines 286-291: This paragraph is the background information of ASNase, so this part should be moved into the Introduction or deleted.

 

Author Response

Reviewer 2

Comments and Suggestions for Authors Shumyantseva et al. reported a study using electroenzymatic model system for the determination of L-asparaginase activity. They found that the substrate-binding can affect the oxidation of the enzyme, thus the electro-oxidation signal can be correlated with the amount of substrate, finally generating the measurement of the Km value of the enzyme. Some flaws should be improved by additional experiments and text revision. Details are below:

1. The design of the experiment in Figure 5 has some problems. Only one point has an Asn concentration below the estimated Km. In enzymatic reaction, lower substrate concentrations will have larger slopes in this curve, therefore more experimental data are needed at low substrate concentrations.

In our paper, we have shown that our results may be useful for the determination of asparaginase activity by means of electrochemical approach and for the screening of L-asparaginase possessing catalytic activity. We plan to work further with asparaginases from different sources, mutant forms of enzyme and to use a range of amino acids. Resulting curve permits to show the influence of substrate L-asparagine on oxidation current of enzyme and evaluate the impact of L-asparagine on oxidation current of protein as measure of catalytic activity.

2. The theory behind Figure 5 and the estimated Km value is not clear. The equation used in curve fitting should be shown in the manuscript.

For the determination of Michaelis constant Km, we used low concentration of substrate in the range of 200 -2500 µM.  The determination of Km was done by fitting experimental curves to obtain this parameter using the Michaelis-Menten model. Km was calculated based on determination of this value as concentration of substrate which produce half of maximum rate (in our case, half of maximum oxidation current). We used the range of concentration as 200, 400, 800, 1600, 2000, 2400 µM.

The dependence presented in Fig. 5 was approximated to a hyperbolic function (R² = 0.968) in accordance with the Michaelis-Menten equation for steady-state enzyme kinetics from the dependences of the catalytic current as a measure of reaction rate on concentration using nonlinear regression analysis [49, 50].

49. Rusling, J.F.; Wang, B.; Yun, S. Electrochemistry of redox enzymes. In Bioelectrochemistry: Fundametals, Experimental Techniques and Applications; Bartlett, P.N., Ed.; John Wiley & Sons, Ltd.: New Jersey, NJ, USA, 2008; pp. 39–85. https://doi.org/10.1002/9780470753842.ch2.
50. Shumyantseva V.V., Bulko T.V., , Koroleva P.I., Shikh E.V., MakhovaA.A., Kisel M.S., Haidukevich I.V., Gilep A.A. Human Cytochrome P450 2C9 and its polymorphic modifications: electroanalysis, catalytic properties, and approaches to the regulation of enzymatic activity. Processes 2022, 10, 383.

We really described our preliminary results concerning the approach for the determination of enzymatic activity of asparaginase by means of electrochemistry as a proof of concept. We plan to continue this investigation as PhD theses using several types of enzymes from different sources, and a range of amino acids.

 

 

3. Line 254: Km was estimated as 400 uM by fitting. How many errors of this value?

Answer

For the determination of Km the values are the means from at least 3 experiments ± S.D. We recalculated the value of Km as 600±70 µM. 

4. Lines 53-60 (References 10-12): These cited methods are not widely accepted methods with broad applications, which are inappropriately cited in the Introduction. These references should be removed. Other methods, such as absorbance, fluorescence, and other convenient and well-established techniques should be cited in this paragraph.

Answer

We agree with Reviewer 2 that methods mentioned in reference 10-12 is not widely accepted methods with broad applications. Electroanalysis is also new approach for these purposes.  However, new methods are developing rapidly, so we prefer to cite these methods for the understanding the role of nanotechnology in enzymology.

Physical, physical and chemical and nanotechnological approaches have also contributed to the development of detection methods for the estimation of enzyme catalytic activity. We mentioned these methods as developing new approaches for the determination of enzyme catalytic activity. We cited “classical” methods such as absorbance, fluorescence for the determination of catalytic activity on p. 1-2 of manuscript (references 1,6,7,8).  

5. The disadvantage and future direction of this method should be discussed in the manuscript.

We added in the manuscript the disadvantage and future direction of this method (p. 11 of manuscript).

 However, the electrochemical approach depends on the type of electrode, electrode modifier and accessibility of substrate to active center of enzyme. Future direction of this method is the construction of new composite materials for the modification of working electrodes with enhanced sensitivity to the electrochemical oxidation of amino acids, investigation of mutant forms of asparaginase from different sources, a range of amino acids as substrates, and inhibitors of this enzyme.

 

6. Line 296: WhyTyr29 is the most important candidate? Is it close to the active site/substrate-binding site? A structure figure illustrating the active site and potential oxidized residues may be helpful.

Tyr29 is a crucial amino acid in the active cite of bacterial L-asparaginases. The recent publication of our colleagues (reference 33. Pokrovskaya, M.V.; Pokrovsky, V.S.; Aleksandrova, S.S.; Sokolov, N.N.; Zhdanov, D.D. Molecular Analysis of L-Asparaginases for Clarification of the Mechanism of Action and Optimization of Pharmacological Functions. Pharmaceutics 2022, 14, 599. https://doi.org/10.3390/pharmaceutics14030599) describes in details the active sites and molecular organization of mechanism of action of L-asparaginases. For this reason, we decided not to repeat the figures of this paper. Tyr25 corresponds to Tyr29 of L-asparaginase from Erwinia carotovora and involved in substrate binding and release of reaction product [38].  We suppose that L-asparagine can change the electrochemical signature at the time of location at the active center on the enzyme by interaction with Tyr29.

7. Line 14, The minus in “0.5-0.6” is incorrectly displayed in the PDF file.

The oxidation peak corresponded to  0.5÷0.6 V. 

8. Lines 92-93: The meaning of “the ability and sensitivity of amino acids of the polypeptide backbone to conformational changes” is unclear. What is the meaning of “the amino acids of the polypeptide backbone”?

We corrected this sentence as

“In our investigation, the ability and susceptibility of the protein to conformational changes registered by electrochemical oxidation were used to estimate the catalytic activity of ASNase.” (p. 4 of manuscript).

 

9. Lines 298-299: The last sentence is incomplete.

We corrected this sentence. Thank you for this comment.

10. Lines 286-291: This paragraph is the background information of ASNase, so this part should be moved into the Introduction or deleted.  

In accordance with Reviewer 2 recommendation, we moved this paragraph into introduction (p. 2 of manuscript).

 

 

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

Shumyantseva et al. report on the development of a method to measure the activity of the Erwininia L-asparaginase (ASNase) based on the electrooxidation of amino acids from the backbone of the ASNase. This electrochemical behavior was obtained by assaying the immobilized enzyme on modified single wall carbon nanotubes as the sensing element. The authors demonstrated that the  oxidation current of the ASNase was reduced in the presence of asparagine in a concentration dependent manner but was unaffected by the presence of the non-substrate glycine. ASNase has been utilized in medicine as a pharmacological antitumor agent and in the food industry in preventing formation of acrylamide in processed foods. Current methods used to measure ASNase activity is based on utilizing with HPLC, circular dichroism, electrophoresis assays, or colorimetric assay measuring presence of substrate asparagine with hydroxylamine or presence of product ammonia with Nessler’s reagent. 

 

Specific Comments:

(1)   This method is effective for examining the activity ASNase for medical and food industry uses. In expanding it use for enzymatic and kinetic utilization, did the authors look at the effect of aspartate or D-asparagine on the electrooxidation properties? 

(2) Are the Michaelis constants reported in this manuscript closer to a binding constant than a Km affinity constant?

(3)   Were the kinetic properties of glutamine determined with this method? 

(4)   I assume the electrochemical method could also be used to look at the effects of competitive inhibitors to asparagine binding. Is this correct?

Author Response

Rev3

Shumyantseva et al. report on the development of a method to measure the activity of the Erwininia L-asparaginase (ASNase) based on the electrooxidation of amino acids from the backbone of the ASNase. This electrochemical behavior was obtained by assaying the immobilized enzyme on modified single wall carbon nanotubes as the sensing element. The authors demonstrated that the  oxidation current of the ASNase was reduced in the presence of asparagine in a concentration dependent manner but was unaffected by the presence of the non-substrate glycine. ASNase has been utilized in medicine as a pharmacological antitumor agent and in the food industry in preventing formation of acrylamide in processed foods. Current methods used to measure ASNase activity is based on utilizing with HPLC, circular dichroism, electrophoresis assays, or colorimetric assay measuring presence of substrate asparagine with hydroxylamine or presence of product ammonia with Nessler’s reagent.    Specific Comments: (1)   

This method is effective for examining the activity ASNase for medical and food industry uses. In expanding it use for enzymatic and kinetic utilization, did the authors look at the effect of aspartate or D-asparagine on the electrooxidation properties? 

Answer

 We thank Reviewer 3 for this suggestion.  We plan to continue this investigation as PhD theses using several types of enzymes from different sources, mutant forms of asparagine and a range of amino acids, such as glutamine, aspartate and D-asparagine. In our present paper, we did not examine aspartate and D-asparagine. The main goal of this paper was to demonstrate suitability of electro analysis for the determination of enzyme activity to substrate L-asparagine.

 

(2) Are the Michaelis constants reported in this manuscript closer to a binding constant than a Km affinity constant?

Thank you for this suggestion. However, it is a complicated question. Tyr29 of L-asparaginase from Erwinia carotovora is involved in substrate binding and release of reaction product  (reference 33. Pokrovskaya, M.V.; Pokrovsky, V.S.; Aleksandrova, S.S.; Sokolov, N.N.; Zhdanov, D.D. Molecular Analysis of L-Asparaginases for Clarification of the Mechanism of Action and Optimization of Pharmacological Functions. Pharmaceutics 2022, 14, 599. https://doi.org/10.3390/pharmaceutics14030599) As we assumed that catalytic activity can be expressed as oxidation current of L-asparaginase, based on Tyr29 electrochemical oxidation, we can determined Michaelis constant. If we assumed that catalytic current is a measure of catalytic activity, we can use the term Michaelis constant Km.  

(3)   Were the kinetic properties of glutamine determined with this method? 

Answer

In our paper, we have shown that L-asparagine and L-glutamine reduce oxidation peak current of asparaginase in different manner, such as L-asparagine - 23%, for Gln - 43%, and Gly - 0%. However, we did not determined kinetic parameters of L-glutamine in this paper. The aim of this paper was to demonstrate applicability of electrochemical approach for the analysis of catalytic activity based on sensitivity of protein to different amino acids. We plan to work with this enzyme in more details, using different types of asparaginases, and a range of amino acids and substrates, such as D-asparagine, succinic acid monoamide and asparaginyl-tRNA.

4)   I assume the electrochemical method could also be used to look at the effects of competitive inhibitors to asparagine binding. Is this correct?

We thank Reviewer 3 for this suggestion. I hope that it is possible. The blocking of binding and catalytic sites of asparaginase must prevent binding with L-asparagine.  We plan to include this investigation in our future work as PhD theses.

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Most of the changes are satisfactory, but two minor changes need to be made:

1. I still suggest removing references 10 and 11. The authors of these references are from the same institute as the authors of this manuscript, but these references do not relate to the topic of the manuscript. Furthermore, the DOI links of references 10 and 11 do not work.

2. Last time I pointed out that there was something wrong with the hyphen in line 14 "0.5-0.6", and the author did not modify it. On my computer, the hyphen on line 14 is wrongly displayed as a sign of division("÷"), while line 198 in "0.5-0.6" is displayed correctly.

Author Response

Dear Dr. Song!

We thank you and reviewer 2 for the additional comments concerning our paper “Electroenzymatic model system for the determination of catalytic activity of Erwinia carotovora L-asparaginase” and understanding the novelty of our research.

Comments and Suggestions for Authors Most of the changes are satisfactory, but two minor changes need to be made:

1. I still suggest removing references 10 and 11. The authors of these references are from the same institute as the authors of this manuscript, but these references do not relate to the topic of the manuscript. Furthermore, the DOI links of references 10 and 11 do not work.
2. Last time I pointed out that there was something wrong with the hyphen in line 14 "0.5-0.6", and the author did not modify it. On my computer, the hyphen on line 14 is wrongly displayed as a sign of division("÷"), while line 198 in "0.5-0.6" is displayed correctly.

 

In accordance of Reviewer 2 recommendation

1. I still suggest removing references 10 and 11. The authors of these references are from the same institute as the authors of this manuscript, but these references do not relate to the topic of the manuscript. Furthermore, the DOI links of references 10 and 11 do not work.

We removed references 10 and 11 in accordance with Reviewer 2 recommendation.  Now manuscript cited 49 appropriate references (highlighted in red) in revised version of paper).

2. Last time I pointed out that there was something wrong with the hyphen in line 14 "0.5-0.6", and the author did not modify it. On my computer, the hyphen on line 14 is wrongly displayed as a sign of division("÷"), while line 198 in "0.5-0.6" is displayed correctly

On line 14 of manuscript, we corrected potential as "0.5-0.6"

Thank you very much for these comments.

Sincerely,

Victoria Shumyantseva, Dr Sci., Prof,

Member of Editorial Board of Processes

 

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The manuscript by Shumyantseva reported a study on the development of an asparaginase detection system using electro-oxidation. Although the idea in this work is new, the study is quite preliminary and lacks sufficient validations for sensitivity, stability, and robustness.

• As shown in Figure 4, a very high concentration of Asn (33 mM) only reduced 77% signal intensity, suggesting the signal has a low correlation with the substate and activity. This results in the poor fitting result in Figure 5, which gave an inaccurate Km value of 400 uM. Figure 5 also shows that the assay is not suitable for the activity measurement of practical ASNase which often have a low micromolar range. Demonstration of the linearity range and sensitivity of the method should be given.
• The glycine was used as a negative control in this study. However, a major side effect during ASNase activity measurement in clinic applications is glutamine. The activity in presence of glutamine as well as glutamic acid and aspartic acid should be measured using the authors’ method.
• Line 284-288, the proposed oxidation sites should be validated by mutagenesis analysis.
• In several places, the authors mentioned the oxidation of the ASNase backbone. Why is the oxidation on the backbone? Meanwhile, the authors assumed oxidation of tyrosine, …, methionine (lines 284-286) which means the oxidation of the sidechain.
• Line 238, I cannot find the inset in Fig. 3A.
• In the Introduction, the authors should mention the development of fluorescent probes for ASNase activity measurement. On the other hand, the long paragraph (lines 53-66) about P450 is unnecessary. This paragraph should be deleted or shortened significantly. The paragraphs in lines 67-93 should be combined and condensed.
• The language should be improved by a professional editing service or a native English speaker.
• Sub-figures should be more compacted in an organization. For example, three sub-figures of Figure 2 are unnecessarily plotted on two pages. They could be organized in one row.

 

Author Response

Rev1

Responses to Review 1 comments

We want to thank Reviewer 1 for careful and detailed analysis of our paper and understanding the novelty of our research. It was hard work, however proposed electrochemical approach is suitable for the screening of asparaginase possessing catalytic activity.

Comments and Suggestions for Authors

The manuscript by Shumyantseva reported a study on the development of an asparaginase detection system using electro-oxidation. Although the idea in this work is new, the study is quite preliminary and lacks sufficient validations for sensitivity, stability, and robustness.

• As shown in Figure 4, a very high concentration of Asn (33 mM) only reduced 77% signal intensity, suggesting the signal has a low correlation with the substate and activity. This results in the poor fitting result in Figure 5, which gave an inaccurate Km value of 400 uM. Figure 5 also shows that the assay is not suitable for the activity measurement of practical ASNase which often have a low micromolar range. Demonstration of the linearity range and sensitivity of the method should be given.

Response.

Our study describes new approach for the determination of asparaginase catalytic activity based on electrochemical oxidation of amino acids. We plan to work in this direction with other types of asparaganases from different sources.  Figure 4 represents the clear response for high concentration of L-Asp for better demonstration of dynamic conformational changes of enzymes during enzyme catalysis. For the determination of Michaelis constant Km, we used low concentration of substrate in the range of 200 -2500 µM.  The determination of Km was done by fitting experimental curves to obtain this parameter using the Michaelis-Menten model. Sensitivity of proposed electrochemical system for L-Asp corresponded to 0.52 10^-5 A/M. Sensitivity towards asparaginase was determined as 0.80±0.07 µA/µg/µl of protein (Fig 3A of manuscript).The oxidation signals were found to be proportional to the protein concentrations. The irreversible nature of electro oxidation was confirmed by means of comparison of the first and second scan of DPV of asaparaginase.  

 

 

• The glycine was used as a negative control in this study. However, a major side effect during ASNase activity measurement in clinic applications is glutamine. The activity in presence of glutamine as well as glutamic acid and aspartic acid should be measured using the authors’ method.

Response.

We conducted experiments with L-Gln (Fig. 1, answers) for comparison the activity of 33 mM L-Asn and 33mM L-Gln. As can be seen from this figure, L-Asn and L-Gln influenced on the peak current of Asparaginase oxidation, and the amplitude in the presence of L-Asn (25±5%) is lower than amplitude in the presence of L-Gln (40±5%) confirming the preference of L-Asn as substrate. This is a pilot project demonstrating electroenzymatic approach for the assessment of catalityc activity of asparaginase. We thank Reviewer 1 for the helpful advices concerning our future work with this very important from medicinal view point enzyme. 

Fig. 1, answers.

 

 

• Line 284-288, the proposed oxidation sites should be validated by mutagenesis analysis.

 

Response.

We discussed the proposed oxidation sites based on earlier published data concerning mutagenic and informatics studies, published earlier [Borek, D.; Jaskólski, M. Sequence analysis of enzymes with asparaginase activity. Acta Biochim. Pol. 2001, 48, 893-902;  Borek, D.; Kozak, M.; Pei, J.; Jaskolski, M. Crystal structure of active site mutant of antileukemic L-asparaginase reveals conserved zinc-binding site. FEBS J. 2014, 281, 111. http://doi: 10.1111/febs.12906; Pokrovskaya, M.V.; Pokrovsky, V.S.; Aleksandrova, S.S.; Sokolov, N.N.; Zhdanov, D.D. Molecular Analysis of L-Asparaginases for Clarification of the Mechanism of Action and Optimization of Pharmacological Functions. Pharmaceutics 2022, 14, 599. https://doi.org/10.3390/pharmaceutics14030599.

We thank Reviewer 1 for the suggestion for such experiments. However, the conductance of mutagenic analysis of asparaginase from Erwinia carotovora needs long time and additional experiments. 

 

• In several places, the authors mentioned the oxidation of the ASNase backbone. Why is the oxidation on the backbone? Meanwhile, the authors assumed oxidation of tyrosine, …, methionine (lines 284-286) which means the oxidation of the sidechain.

Response.

Electrochemical oxidation may utilize all amino acids of protein assessable for electrochemical oxidation. We changed this term as “electroactivity of protein amino acids residues”  

In conclusion we changed this term as “Electroactivity of protein amino acids residues was used as a measuring tool”.

 

• Line 238, I cannot find the inset in Fig. 3A.

Response.

Sorry for this typo. We used this figure as Fig. 3C.

•  
• In the Introduction, the authors should mention the development of fluorescent probes for ASNase activity measurement. On the other hand, the long paragraph (lines 53-66) about P450 is unnecessary. This paragraph should be deleted or shortened significantly. The paragraphs in lines 67-93 should be combined and condensed.

Response.

Thank you this comments. We deleted long paragraph concerning cytochrome P450 and condensed the paragraphs in lines 67-93

 

• The language should be improved by a professional editing service or a native English speaker.

Response.

We thank Reviewer 1 for this comments. We tried to improve our English language.  The manuscript was subjected to English editing by American Journal Experts editing service and the changes are tracked. 

 

• Sub-figures should be more compacted in an organization. For example, three sub-figures of Figure 2 are unnecessarily plotted on two pages. They could be organized in one row.

Response.

We improves organization of our paper and organized Fig 2 (A, B, C) in one row. 

 

Author Response File: Author Response.docx

Reviewer 2 Report

Page 2, line 56, please replace « house » by « horse »

Page 3, line 135, please replace “oC” bu “°C”. Please explain how the electrode was kept at 37°C during the incubation time. Was the electrode rinsed after the incubation, or the electrochemical measurement is done in the presence of the substrate?

In figure 2b, the unit of v1/2 is V1/2 s -1/2, not V1/2 s1/2

Page 4, line 168, please replace Fig 2B by Fig 2c, because Figure 2B is about the variation of peak intensity with square root of scan rate. Please comment this figure and say that this relationship is valid for a diffusion controlled redox process.

Page 4, line 171, please indicate how the calculation of the specific surface area were performed. What reference value of the charge was used?

For figure 3, please rewrite the legend because it is not clear just by suing (-), (--). Please indicate what are the colors used for!

 

Page 8, line 238, there is not inset of Figure 3A!!!!!

Page 9, please make more comments on the difference between the obtained Km constant by the electrochemical method compared to Nessler method.

Page 11, line 276, please change the character between Asp90 and Lys162 to a Latin character.

Author Response

Responses to Review 2 comments

We want to thank Reviewer 1 for careful and detailed analysis of our paper and understanding the novelty of our research. It was hard work, however proposed electrochemical approach is suitable for the screening of asparaginase possessing catalytic activity.

 

Rev 2.

Comments and Suggestions for Authors

Page 2, line 56, please replace « house » by « horse »

Response.

Thank you for this comments. The typo was corrected.  

Page 3, line 135, please replace “oC” bu “°C”. Please explain how the electrode was kept at 37°C during the incubation time. Was the electrode rinsed after the incubation, or the electrochemical measurement is done in the presence of the substrate?

Response

For substrate binding we performed experiments at room temperature. Asparaginase was incubated with L-asparagine 5 min, and then DPV measurements were performed.  

In figure 2b, the unit of v1/2 is V1/2 s -1/2, not V1/2 s1/2

Response

We corrected Fig.2B, unit on axis as n^1/2, (V/s)^1/2

 

Page 4, line 168, please replace Fig 2B by Fig 2c, because Figure 2B is about the variation of peak intensity with square root of scan rate. Please comment this figure and say that this relationship is valid for a diffusion controlled redox process.

Response

We corrected these figures in accordance with Reviewer 2 comments. We added explanation concerning the diffusion controlled redox process for external electrolyte ferri/ferrro cyanide Fe(CN)₆ ^3-/4-  .

“The linear dependence of the peak current versus the square root of the scan rate was found and confirmed a diffusion-controlled process (Fig. 2B)”.

 

Page 4, line 171, please indicate how the calculation of the specific surface area were performed. What reference value of the charge was used?

Response

We described the calculation of specific surface area of screen-printed electrodes SPE and modified SPE  in detail earlier in Sigolaeva, L.V.; Bulko, T.V.; Kozin, M.S.; Zhang, W.; Köhler, M.; Romanenko, I.; Yuan, J.; Schacher, F.H.; Pergushov, D.V.; Shumyantseva, V.V. Long-term stable poly(ionic liquid)/MWCNTs inks enable enhanced surface modification for electrooxidative detection and quantification of dsDNA. Polymer 2019, 168, 95–103. https://doi.org/10.1016/j.polymer.2019.02.005. For this reason in this paper we used the same approach and electrochemical parameters for the calculation of specific surface area with reference for corresponding papers.

 

Polymer 2019, 168, 95–103.

As seen from the Figure 2 (A, B) , the anodic/cathodic peak currents exhibit a linear relationship with the square root of the scan rate in the range of 10–100 mV/s, providing evidence for a chemically reversible redox process. The electroactive surface area, A (cm²), of the electrode can be defined using the Randles–Sevcik equation expressed by the following equation:

                                           (1),

where I_p is the current maximum in amps, A is the electroactive surface area in cm², D is the diffusion coefficient in cm²/s, ν is the scan rate in V/s, , C is the concentration in mol/cm³, and n is the number of electrons transferred in the redox event. By substituting the known values of D = 7.6 × 10⁻⁶cm²/s [ Chen, H.C., Chang, C.C., Yang, K.H., Mai, F.D., Tseng, C.L., Chen, L.Y., Hwang, B.J. and Liu, Y.C. (2018). Polypyrrole electrode with a greater electroactive surface electrochemically polymerized in plasmon-activated water. Journal of the Taiwan Institute of Chemical Engineers 82, 252-260], n = 1, and C = 0.005 M for the used K₃[Fe(CN)₆]/K₄[Fe(CH)₆] redox probe into the equation (1), the surface areas of each electrode modification can easily be extracted from the slope with the dependence of I_p vs ν^1/2.

For figure 3, please rewrite the legend because it is not clear just by suing (-), (--). Please indicate what are the colors used for!

 Response

We corrected Figure legends as

Figure 3. (A). DPV electrochemical oxidation signals of SPE/SWCNT/ASNase, 3 µg/µl (-), and 5 µg/µl (-), SPE/SWCNT (---). (B). DPVs of the first (-) and the second (---) scan of SPE/SWCNT/ASNase (5 µg/µl). (C) DPVs of SPE/SWCNT/ASNase, 3 µg/µl (-), 33 mM L-Asn (-), and 33 mM Gly (-) on SPE/SWCNT, SPE/SWCNT (---).  Supporting electrolyte: 0.1 M potassium-phosphate buffer containing 0.05 M NaCl, pH 7.4.

 

 

Page 8, line 238, there is not inset of Figure 3A!!!!!

Response

Sorry for this typo. We used this figure as Fig. 3C.

Page 9, please make more comments on the difference between the obtained Km constant by the electrochemical method compared to Nessler method.

Response

Thank you for this suggestion.

Electrochemical approach for the estimation of catalytic activity of asparaginase was proposed based on electro oxidation of electroactive amino acids of asparaginase. This approach permits to calculate Km of substrate L-Asn. We obtained this value with the same order of magnitude as Km determined by Nessler method. The approximately three 3 time difference may be dealing with the imperfection of both approaches. The most commonly used Nessler reaction requires the use of highly toxic reagents with poor stability, Nessler reaction has low reproducibility, complicated operations, and are not suitable for the detection of clinical samples [Res. Chem. 2021, 3, 100103. https://doi.org/10.1016/j.rechem.]. Fluorescence analysis method needs synthesis of fluorogenic probe for the assessment of catalytic activity of enzyme. From this viewpoint, electrochemical approach is the most robust and effective. However, electrochemical approach depends on the type of electrode, electrode modifier and accessibility of substrate to enzyme active center.  For these reasons the difference between Km value obtained by means of Nessler reaction and electrochemical approach may be registered.

We added discussion concerning Km value in our manuscript.  

 

 

Page 11, line 276, please change the character between Asp90 and Lys162 to a Latin character.

Response

We rewrites this sentence as “We assumed that amino acids with ability for electrochemical oxidation, such as Tyr, Trp, His, Cys, Cys-cys and Met [21-29] are responsible for the response on substrate interaction.

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

This manuscript describes the development of an electrochemical method for the determination of  activity of L-asparaginase. It is well written and the result makes sound.  I have no special comments.

Reviewer 4 Report

 

The reviewed paper by Viktoria Shumyantseva et al. presents an electrochemical method for the direct determination of the catalytic activity of L-asparaginase. The proposed procedure is based on the voltammetrically followed electrooxidation of amino acids from L-asparaginase polypeptide backbones.

The manuscript is well written and easy to follow. The experimental part describes electrochemical studies adequately, and the results are convincing. Also, the conclusions derived stick to the outcomes of the work. My single criticism concerns the use of glycine only as of the concurrent substrate for Asn. Why not any other amino acid tried? It could widen the scope of the method.

Anyhow, I consider the paper suitable for publication.

Round 2

Reviewer 1 Report

The revised manuscript has not been improved significantly. Several major problems indicated in my previous comments were not well solved in the  revised manuscript. So I cannot suggest publishing it.