Merits of HPLC-based method over spectrophotometric method for assessing the kinetics and inhibition of mammalian adenosine deaminase.
Measurement of adenosine deaminase (ADA) activity using spectrophotometric method presents problem, regarding the quantitative estimation of the substrate degradation and product formation, due to the closely apposed lambda(max) of the substrates, product and the inhibitor. The feasibility of applying reverse-phase HPLC technique, for studying adenosine deaminase-catalyzed reaction product and inhibition study was examined. We have drawn a comparison between the HPLC-based method over the corresponding spectrophotometric method. A gradient elution pattern was used to separate substrate (adenosine and deoxyadenosine), product (inosine and deoxyinosine) and standard adenosine deaminase inhibitor (erythro-9-(3-nonyl-p-aminobenzyl)-adenine) in the HPLC method. The product formation was quantitated by monitoring the absorbance at 260 nm with the progress of time. The limit of detection as well as the limit of quantification of the respective enzymatic product were found to be in nano molar (nM) range in the HPLC method. This study was also extended to monitor adenosine deaminase activity in different cancer cells of hematological origin. The HPLC-based method is found to be suitable for the quantitative estimation of adenosine deaminase-catalyzed reaction product and for studying inhibition mechanism of different inhibitors. The HPLC-based method has specific advantages over the spectrophotometric method. Moreover, the concentration of different nucleotides in cell lysate and body fluid can be measured using this HPLC method.
Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S Nagar, Mohali, Punjab 160062, India.
Manash K
MK
Paul
Vikas
V
Grover
Anup K
AK
Mukhopadhyay
D000243:Adenosine Deaminase / Q000378:metabolism*; D058892:Adenosine Deaminase Inhibitors; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007700:Kinetics; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D058892:Adenosine Deaminase Inhibitors; D000243:Adenosine Deaminase