title: Miniaturization of hydrolase assays in thermocyclers.
abstract: We adapted the protocols of reducing sugar measurements with dinitrosalicylic acid and bicinchoninic acid for thermocyclers and their use in enzymatic assays for hydrolases such as amylase and β-1,3-glucanase. The use of thermocyclers for these enzymatic assays resulted in a 10 times reduction in the amount of reagent and volume of the sample needed when compared with conventional microplate protocols. We standardized absorbance readings from the polymerase chain reaction plates, which allowed us to make direct readings of the techniques above, and a β-glycosidase assay was also established under the same conditions. Standardization of the enzymatic reaction in thermocyclers resulted in less time-consuming temperature calibrations and without loss of volume through leakage or evaporation from the microplate. Kinetic parameters were successfully obtained, and the use of the thermocycler allowed the measurement of enzymatic activities in biological samples from the field with a limited amount of protein.
authors.affiliation: National Institute of Metrology, Quality, and Technology (INMETRO), Rio de Janeiro 20261-232, Brazil; Oswaldo Cruz Institute (IOC, FIOCRUZ), Rio de Janeiro 21040-900, Brazil.
authors.forename: Severino A
authors.initials: SA
authors.lastname: Lucena
authors.forename: Caroline S
authors.initials: CS
authors.lastname: Moraes
authors.forename: Samara G
authors.initials: SG
authors.lastname: Costa
authors.forename: Wanderley
authors.initials: W
authors.lastname: de Souza
authors.forename: Patrícia
authors.initials: P
authors.lastname: Azambuja
authors.forename: Eloi S
authors.initials: ES
authors.lastname: Garcia
authors.forename: Fernando A
authors.initials: FA
authors.lastname: Genta
mesh_terms: D000681:Amylases / Q000378:metabolism*; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods*; D043326:Glucan 1,3-beta-Glucosidase / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008904:Miniaturization / Q000295:instrumentation*; D011804:Quinolines / Q000737:chemistry; D012459:Salicylates / Q000737:chemistry; D012463:Saliva / Q000201:enzymology; D013213:Starch / Q000378:metabolism
chemical_list: D011804:Quinolines; D012459:Salicylates; D013213:Starch; C047117:bicinchoninic acid; D000681:Amylases; D043326:Glucan 1,3-beta-Glucosidase