Colorimetric method for enzymatic screening assay of ATP using Fe(III)-xylenol orange complex formation.
In hygiene management, recently there has been a significant need for screening methods for microbial contamination by visual observation or with commonly used colorimetric apparatus. The amount of adenosine triphosphate (ATP) can serve as the index of a microorganism. This paper describes the development of a colorimetric method for the assay of ATP, using enzymatic cycling and Fe(III)-xylenol orange (XO) complex formation. The color characteristics of the Fe(III)-XO complexes, which show a distinct color change from yellow to purple, assist the visual observation in screening work. In this method, a trace amount of ATP was converted to pyruvate, which was further amplified exponentially with coupled enzymatic reactions. Eventually, pyruvate was converted to the Fe(III)-XO complexes through pyruvate oxidase reaction and Fe(II) oxidation. As the assay result, yellow or purple color was observed: A yellow color indicates that the ATP concentration is lower than the criterion of the test, and a purple color indicates that the ATP concentration is higher than the criterion. The method was applied to the assay of ATP extracted from Escherichia coli cells added to cow milk.
Ishida
Akihiko
A
Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, Nishi 8, Kita 13, Sapporo, 060-8628, Japan. ishida-a@eng.hokudai.ac.jp
Yamada
Yasuko
Y
Kamidate
Tamio
T
D000255:Adenosine Triphosphate / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002417:Cattle; D002460:Cell Line; D003124:Colorimetry / Q000379:methods*; D004926:Escherichia coli / Q000166:cytology / Q000472:pathogenicity; D005290:Ferric Compounds / Q000737:chemistry*; D005296:Ferrous Compounds / Q000737:chemistry; D008892:Milk / Q000737:chemistry*; D009942:Organometallic Compounds / Q000737:chemistry*; D010084:Oxidation-Reduction; D010636:Phenols; D011771:Pyruvate Oxidase / Q000378:metabolism; D013454:Sulfoxides; D014992:Xylenes / Q000737:chemistry*
D005290:Ferric Compounds; D005296:Ferrous Compounds; D009942:Organometallic Compounds; D010636:Phenols; D013454:Sulfoxides; D014992:Xylenes; D000255:Adenosine Triphosphate; D011771:Pyruvate Oxidase; C016833:xylenol orange