New method for guanase activity measurement by high-performance liquid chromatography.
A rapid isocratic high-performance liquid chromatographic method for the determination of guanase (EC 3.5.4.3) activity is proposed. The method is highly reproducible, with a coefficient of variation of less than 1%, and requires only ca. 10 min for a complete chromatographic separation of the enzyme reaction mixture. The method allows the detection of nanomolar changes in the concentrations of both the substrate and the product, and does not require additional reactions or sample pretreatment. Kinetic studies with the proposed method showed the guanase activity to have an apparent Michaelis constant of 13.3 and 8.5 microM, and a maximum rate of 1.95 and 3.84 pmol/min per mg protein at 37 degrees C, in Tris-HCl and phosphate buffer, respectively.
Dipartimento di Chimica, Università degli Studi di Roma, La Sapienza, Italy.
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S
Canepari
V
V
Carunchio
A M
AM
Girelli
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A
Messina
D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D006147:Guanine / Q000032:analysis / Q000378:metabolism; D006148:Guanine Deaminase / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D013056:Spectrophotometry, Ultraviolet; D014970:Xanthines / Q000032:analysis
D002021:Buffers; D007202:Indicators and Reagents; D014970:Xanthines; D006147:Guanine; D006148:Guanine Deaminase