Methods and kits for quantifying adenosine-containing molecules

A method for quantifying adenosine-containing compounds within an aqueous composition comprising the steps of:

• a) providing a fluorescent probe comprising:
• i) an adenosine scaffold comprising S-adenosyl homocysteine (AdoHcy), and
• ii) a 5-carboxytetramethylrhodamine (TAMRA) fluorophore;
• wherein the 5-carboxyl functional group within the TAMRA fluorophore is covalently bound to the α-amino moiety within AdoHcy;
• b) providing a mutated Helicobacter pylori (Hp) 5′-methylthioadenosine/S-adenosyl homocysteine nucleosidase (MTAN enzyme), wherein the mutated MTAN enzyme comprises an amino acid sequence selected from the group consisting of:
• i) SEQ ID NO:
• 4, wherein the cysteine residue at position 12 is conjugated to a secondary fluorophore,
• ii) SEQ ID NO:
• 6, wherein the cysteine residue at position 213 is conjugated to a secondary fluorophore, and
• iii) SEQ ID NO:
• 8, wherein the amino acid residue at one or both of positions 12 and 213 is a cysteine residue conjugated to a secondary fluorophore, and
• wherein the secondary fluorophore consists of DABCYL-maleimide or C5-maleimide;
• wherein the secondary fluorophore conjugated to any of the mutated MTAN enzymes comprises a fluorescently-active moiety and a thiol-reactive crosslinker that bridges the fluorescently-active moiety to the cysteine residue;
• c) forming a protein-probe complex between the mutated MTAN enzyme and the fluorescent probe;
• d) measuring the fluorescence intensity of the fluorescent probe within the protein-probe complex;
• e) combining an adenosine-containing compound, the adenosine-containing compound selected from the group consisting of AdoHcy, 5′-methylthioadenosine, adenosine diphosphate, adenosine monophosphate, and adenosine, with the protein-probe complex to form an adenosine quantification mixture;
• f) measuring the increase of fluorescence intensity of the fluorescent probe in the presence of the adenosine quantification mixture;
• and
• g) calculating the amount of the adenosine-containing compound within the adenosine quantification mixture using the increase of fluorescence intensity of the fluorescent probe in the presence of the adenosine quantification mixture.