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Updated: Feb 16, 2026
Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics
Published on: October 13, 2020
False positives in using the zymogram assay for identification of peptidoglycan hydrolases
Cristian A Escobar1, Timothy A Cross2
1Institute of Molecular Biophysics, and National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310 USA.
Abstract:
Zymogram assays have been used extensively to identify novel peptidoglycan hydrolases. In this study it is reported that the zymogram is susceptible to false positive results when highly positively charged proteins are assayed. As an example, we report on the case of the ChiZ membrane protein from the Mycobacterium tuberculosis divisome, which previously was described as a peptidoglycan hydrolase. Even though the full length ChiZ protein was able to produce positive assay results, other direct methods for measuring peptidoglycan hydrolysis do not provide convincing evidence that ChiZ has peptidoglycan hydrolysis activity. We show that the false positive result is produced by the highly positively charged N-terminal region of ChiZ. Thus, we developed a zymogram control that can be used to identify false positives results. This control assay lacks the refolding step in the normal zymogram assay. For lysozyme the control assay shows no activity, while the N-terminal region of ChiZ shows a false positive result. Given the limitations of the zymogram assay to reliably identify peptidoglycan hydrolases, we recommend using the zymogram control assay together with other methods to evaluate possible peptidoglycan hydrolysis activity.
Insights
Zymogram assays can yield false positives for peptidoglycan hydrolases due to highly charged proteins. A new control assay helps identify these false results, improving enzyme activity analysis.
Area of Science:
- Microbiology
- Biochemistry
- Enzymology
Background:
- Zymogram assays are common for identifying peptidoglycan hydrolases.
- Concerns exist regarding the reliability of zymogram assays for certain proteins.
Purpose of the Study:
- To investigate false positive results in zymogram assays for peptidoglycan hydrolases.
- To evaluate the activity of the Mycobacterium tuberculosis ChiZ protein.
- To develop a control assay for zymogram analysis.
Main Methods:
- Zymogram assays were performed on the ChiZ protein.
- Direct methods were used to measure peptidoglycan hydrolysis.
- A modified zymogram control assay lacking a refolding step was developed.
Main Results:
- The full-length ChiZ protein showed positive zymogram results, but direct assays did not confirm hydrolysis activity.
- The N-terminal region of ChiZ was identified as the cause of false positive zymogram results.
- The control assay showed no activity for lysozyme but a false positive for the ChiZ N-terminal region.
Conclusions:
- Zymogram assays are susceptible to false positives with highly charged proteins like ChiZ.
- A zymogram control assay can help identify false positive results.
- Combined use of zymogram control and direct methods is recommended for accurate peptidoglycan hydrolase identification.
