title: Amino acids interference on the quantification of reducing sugars by the 3,5-dinitrosalicylic acid assay mislead carbohydrase activity measurements.
abstract: This study evaluated the interference of the amino acids tryptophan, cysteine, histidine, tyrosine, hydroxyproline, leucine, proline, serine, glycine, valine, glutamic acid, phenylalanine, and methionine on the measurement of reducing sugars using a phenol-free 3,5-dinitrosalicylic acid (DNS) reagent. It was found that in reaction mixtures containing 20mM of either tryptophan, cysteine, histidine, tyrosine, or hydroxyproline the measurement of 3.7 mM glucose was overestimated by 76%, 50%, 35%, 18%, and 10%, respectively. The amino acids valine, glutamic acid, and phenylalanine did not affect the DNS reaction, while methionine decreased the color development by 5%. The measurement of glucose, xylose, arabinose, and cellobiose at the 3.7-12.4 mM range in the presence of 20 mM cysteine resulted in an overestimated concentration of 34.8-50%. Enzymatic assays for measuring xylanolytic and filter paper activity (FPAse) were conducted in the presence of 20-60 mM cysteine, and compared to cysteine-free assays. In the presence of cysteine, the measured xylanase activity increased threefold and the FPAse activity increased twofold due to the overestimation of the reducing sugar concentrations in the assays. The interference from cysteine was reduced to a maximum of 8.6% when a DNS reagent containing phenol was used.
authors.affiliation: Federal University of Rio de Janeiro, Chemistry Institute, Avenida Athos da Silveira Ramos, 149 Centro de Tecnologia, Bloco A, Cidade Universitaria, CEP: 21941-909 Rio de Janeiro, RJ, Brazil.
authors.forename: Ricardo Sposina Sobral
authors.initials: RS
authors.lastname: Teixeira
authors.forename: Ayla Sant'Ana
authors.initials: AS
authors.lastname: da Silva
authors.forename: Viridiana Santana
authors.initials: VS
authors.lastname: Ferreira-Leitão
authors.forename: Elba Pinto
authors.initials: EP
authors.lastname: da Silva Bon
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D016477:Artifacts; D002241:Carbohydrates / Q000737:chemistry*; D003124:Colorimetry; D057075:Enzyme Assays / Q000379:methods*; D006026:Glycoside Hydrolases / Q000032:analysis / Q000378:metabolism*; D019163:Reducing Agents / Q000737:chemistry* / Q000378:metabolism; D012459:Salicylates / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D002241:Carbohydrates; D019163:Reducing Agents; D012459:Salicylates; C027011:3,5-dinitrosalicylic acid; D006026:Glycoside Hydrolases; C019822:carbohydrase