title: Carbonic anhydrase inhibitors: the very weak inhibitors dithiothreitol, beta-mercaptoethanol, tris(carboxyethyl)phosphine and threitol interfere with the binding of sulfonamides to isozymes II and IX.
abstract: The inhibition of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) with dithiothreitol, 2-mercaptoethanol, tris(carboxyethyl)phosphine (reducing agent frequently added to enzyme assay buffers) and threitol has been investigated. The agents were very weak inhibitors of isozymes CA II and CA IX, but unexpectedly, strongly influenced the binding of the low nanomolar sulfonamide inhibitor acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide). Acetazolamide affinity for all investigated CAs diminished orders of magnitude with increasing concentrations of these agents in the assay system. DTT and similar derivatives should not be added to the assay buffers used in monitoring CA activity/inhibition, as they lead to under-estimation of the binding constants, by a mechanism probably involving the formation of ternary complexes.
authors.affiliation: Universita degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Room 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy.
authors.forename: Alessio
authors.initials: A
authors.lastname: Innocenti
authors.forename: Mika
authors.initials: M
authors.lastname: Hilvo
authors.forename: Andrea
authors.initials: A
authors.lastname: Scozzafava
authors.forename: Mikaela
authors.initials: M
authors.lastname: Lindfors
authors.forename: Henri R
authors.initials: HR
authors.lastname: Nordlund
authors.forename: Markku S
authors.initials: MS
authors.lastname: Kulomaa
authors.forename: Seppo
authors.initials: S
authors.lastname: Parkkila
authors.forename: Claudiu T
authors.initials: CT
authors.lastname: Supuran
mesh_terms: D000086:Acetazolamide / Q000378:metabolism*; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000378:metabolism; D024402:Carbonic Anhydrase II / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000071231:Carbonic Anhydrase IX; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology*; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes; D008623:Mercaptoethanol / Q000494:pharmacology*; D015394:Molecular Structure; D010720:Phosphines / Q000494:pharmacology*; D013053:Spectrophotometry; D013402:Sugar Alcohols / Q000494:pharmacology*
chemical_list: D000086:Acetazolamide; D000951:Antigens, Neoplasm; D024402:Carbonic Anhydrase II; D000071231:Carbonic Anhydrase IX; D002257:Carbonic Anhydrase Inhibitors; D002256:Carbonic Anhydrases; D004229:Dithiothreitol; D007527:Isoenzymes; D008623:Mercaptoethanol; D010720:Phosphines; D013402:Sugar Alcohols; C080938:tris(2-carboxyethyl)phosphine; C003460:threitol; C089226:CA9 protein, human