ISSN print edition: 0366-6352 ISSN electronic edition: 1336-9075 Registr. No.: MK SR 9/7 Published monthly

The exploitation of a pH-sensitive fluorescent probe for monitoring of carbonic anhydrase activity in vitro under weakly acidic conditions

Radovan Murín, Roman Eibner, Jakub Joniak, Karin Schniererová, Mária Dobšovičová, Vladimír Garaj, Jaroslav Galba, Tatiana Matáková, Henrieta Stankovičová, and Jakub Šofranko

Department of Medical Biochemistry, Jessenius Faculty of Medicine in Martin, Comenius University Bratislava, Martin, Slovakia

E-mail: murin@jfmed.uniba.sk

Received: 24 February 2025  Accepted: 11 July 2025

Abstract:

Carbonic anhydrases (CAs) are essential enzymes that maintain pH balance within cellular compartments and the tumor microenvironment. Altered expression of CA family genes is associated with glioblastoma progression, highlighting the need for accurate assessment of CA activity under varying pH conditions. This study evaluates CA activity in human glioblastoma T98G cell lysates under acidic conditions. Classical chromogenic assays based on the hydrolysis of p-nitrophenol derivatives are effective primarily in basic pH ranges. To enhance the detection of CA activity in acidic environments, we applied small-molecule fluorescent “turn-on” pH probes based on 3-(dimethylamino)coumarin and 2-(dimethylamino)benzo[f]coumarin skeletons. The selected probe demonstrated high sensitivity for monitoring proton level changes between pH 5 and 7, enabling the assessment of CA activity under acidic conditions. Acetazolamide, a CA inhibitor, nearly completely inhibited CA activity at acidic pH levels when applied at a concentration of 200 µM, significantly surpassing its inhibitory capacity at pH 8.2. While chromogenic assays effectively measure CA activity under alkaline conditions, the fluorescent probe enables detection of CA activity in acidic environments. The observed pH-dependent effect of acetazolamide on CA activity suggests that the novel assay could be beneficial for reassessing the impact of CA inhibitors under acidic conditions, which are characteristic of tumor microenvironments. Although, the assay dispenses to a possibility to exploit fluorescence sensitive probes to estimate CA activity not only at acidic conditions.

Keywords: EC 4.2.1.1 (carbonic anhydrases); Acetazolamide; Fluorometry; Enzymatic assay

Full paper is available at www.springerlink.com.

DOI: 10.1007/s11696-025-04252-9

Chemical Papers 79 (10) 7127–7137 (2025)