A sensitive assay for maleimide groups.

R Singh

doi:10.1021/bc00028a011

Home/Archive/PubMed

PubMed · 7948101

A sensitive assay for maleimide groups.

R Singh

View original source ↗Export metadata ↓

Abstract

A sensitive spectrophotometric assay has been developed for maleimide groups incorporated into proteins. The assay involves the reaction of maleimide with an excess of cysteine and quantitation of the remaining cysteine using an enzymatic assay that is based on the stoichiometric conversion of an inactive disulfide form of papain to the active enzyme. The restored enzymatic activity is then measured using a chromogenic substrate. The amount of maleimide is calculated as the difference between the amounts of initial cysteine and assayed remaining cysteine. The limits of detection of this assay are about 0.1 nmol maleimide in a final assay volume of 1.2 mL. This assay is about 100-fold more sensitive than a similar assay using Ellman's reagent.

Explore related subjects

Cysteine Dithionitrobenzoic Acid Ethylmaleimide Kinetics Maleimides Papain Proteins Journal Article

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

R Singh. A sensitive assay for maleimide groups.. https://doi.org/10.1021/bc00028a011

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2026

Crosstalk between cysteine and lysine modifications: Integrating redox and metabolic regulation.

Protein post-translational modifications (PTMs) on amino acid residues enable dynamic cellular responses to changes in metabolic and redox state. Cysteine and lysine are among the most extensively modified amino acid residues, with both undergoing a diversity of acylation and oxidative modifications. Indeed, proximal (<10 Å) cysteine and lysine residues may form integration nodes for crosstalk between metabolism and redox homeostasis pathways. This review highlights the interaction of proximal Cys-Lys residues, including influence on residue pKa by local electrostatics, cysteine-to-lysine transfer of PTM moieties, and covalent crosslinking. We discuss candidate Cys-Lys regulatory pairs in proteins involved in redox regulation, proteostasis, metabolic adaptation and inflammation. We further utilize computational modeling to identify proximity between cysteine and lysine residues in proteins known to be regulated by acylation and oxidative PTMs, and to demonstrate changes in these distances and local electrostatic potential due to lysine acetylation. Finally, we review how mass spectrometry-based proteomics and machine-learning PTM predictive tools can enable the identification, validation, and interpretation of proximal Cys-Lys interactions that regulate cellular responses to oxidative challenge and metabolic flux.

Cysteine↗

PubMed2026

Analysis of Confounding Factors in Reactive Cysteine Profiling Reveals Enhanced Chromatin-Protein Association via CDK7 Inhibition by THZ1.

Recent advances in activity-based proteome profiling (ABPP) have enabled the global mapping of cysteine ligandability, uncovering novel biological insights and opportunities for identifying disease vulnerabilities. While both live-cell-based and native-lysate-based ABPP have been applied, how cysteine ligandability differs between these systems and what factors influence these measurements remain unclear. Building on our previous development of a high-throughput TMT-ABPP workflow for native lysates, here we adapt the protocol for live cells and systematically compare cysteine ligandability across both platforms. Our analysis reveals three major contributors to the discrepancies: in-cellular cysteine accessibility, protein abundance changes, and protein relocalization. Notably, we highlight that the CDK7 inhibitor THZ1 induces substantial protein relocalization and promotes chromatin binding. Together, these results provide a practical framework for ABPP experimental design and data interpretation, supporting the more accurate application of ABPP in functional proteomics and drug discovery.

Cysteine↗

PubMed2022

S-nitrosylation of CSF1 receptor increases the efficacy of CSF1R blockage against prostate cancer.

Sustained oxidative stress in castration-resistant prostate cancer (CRPC) cells potentiates the overall tumor microenvironment (TME). Targeting the TME using colony-stimulating factor 1 receptor (CSF1R) inhibition is a promising therapy for CRPC. However, the therapeutic response to sustained CSF1R inhibition (CSF1Ri) is limited as a monotherapy. We hypothesized that one of the underlying causes for the reduced efficacy of CSF1Ri and increased oxidation in CRPC is the upregulation and uncoupling of endothelial nitric oxide synthase (NOS3). Here we show that in high-grade PCa human specimens, NOS3 abundance positively correlates with CSF1-CSF1R signaling and remains uncoupled. The uncoupling diminishes NOS3 generation of sufficient nitric oxide (NO) required for S-nitrosylation of CSF1R at specific cysteine sites (Cys 224, Cys 278, and Cys 830). Exogenous S-nitrosothiol administration (with S-nitrosoglutathione (GSNO)) induces S-nitrosylation of CSF1R and rescues the excess oxidation in tumor regions, in turn suppressing the tumor-promoting cytokines which are ineffectively suppressed by CSF1R blockade. Together these results suggest that NO administration could act as an effective combinatorial partner with CSF1R blockade against CRPC. In this context, we further show that exogenous NO treatment with GSNOR successfully augments the anti-tumor ability of CSF1Ri to effectively reduce the overall tumor burden, decreases the intratumoral percentage of anti-inflammatory macrophages, myeloid-derived progenitor cells and increases the percentage of pro-inflammatory macrophages, cytotoxic T lymphocytes, and effector T cells, respectively. Together, these findings support the concept that the NO-CSF1Ri combination has the potential to act as a therapeutic agent that restores control over TME, which in turn could improve the outcomes of PCa patients.

Cysteine↗