Direct measurement of enzyme activity with infrared spectroscopy.
A direct approach to enzyme activity measurements is presented. Vibrational spectroscopy can monitor the progress of enzymatic reactions because the vibrational spectrum of substrates and products usually differs. This is demonstrated by the example of ATP hydrolysis by Ca(2+)-ATPase: The substrate concentration can be followed using the infrared absorption of the alpha- and beta-PO(2)(-) phosphate groups of ATP, and the product concentration can be followed using the PO(3)(2-) absorption of P(i) and of the beta-phosphate of ADP. The results of the infrared spectroscopic measurement of ATPase activity and of an independent activity assay agree very well. The main advantage of the infrared method is that it observes the reaction of interest directly--that is, no activity assay that converts the progress of the reaction into an observable quantity is required.
Thoenges
Detlef
D
Institut für Biophysik, Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
Barth
Andreas
A
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000032:analysis; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011817:Rabbits; D013055:Spectrophotometry, Infrared / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared; D013379:Substrate Specificity
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases