Detection of histidine kinases via a filter-based assay and reverse-phase thin-layer chromatographic phosphoamino acid analysis

• Published in: Analytical biochemistry Vol. 323; no. 1; pp. 122 - 126
• Main Authors: Tan, Eiling, Lin Zu, Xin, Yeoh, George C, Besant, Paul G, Attwood, Paul V
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2003
• Subjects: Acid-labile; Alkali-stable; Chromatography, Thin Layer - methods; Fungal Proteins - analysis; Histidine - analogs & derivatives; Histidine - analysis; Histidine Kinase; Histones - analysis; Kinase assay; Membranes, Artificial; Phosphohistidine; Phosphorylation; Protein Kinases - analysis; Protein Kinases - metabolism; Reverse-phase thin-layer chromatography; Reverse-phase thin-layer chromatography; Alkali-stable; Kinase assay; Phosphohistidine; Acid-labile
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2003.08.035

The methods that detect histidine phosphorylation have largely been either laborious or difficult to apply quantitatively. The major difficulty in assessing for its presence is its alkali-stable, acid-labile nature. While an assay that detects alkali-stable phosphorylation has been developed, it does not distinguish phosphohistidine from other alkali-stable phosphoamino acids. Using this established method, we extend the assay to facilitate the specific detection of phosphohistidine. We use the acid-lability of phosphohistidine as a defining feature in our approach for its detection. In addition, reverse-phase thin-layer chromatography was utilized to conclusively demonstrate the viability of the conditions that we implement in the assay for the selective detection of phosphohistidine. In summary, this report describes a rapid filter-based kinase assay that quantitatively measures histidine kinase activity, even in the presence of tyrosine kinase activity.