Determination of beta-amylase activity by a fluorimetric 2-p-toluidinylnaphthalene-6-sulfonate flow-injection analysis (2, 6-TNS-FIA) method, using amylose and amylopectin as substrates.
2-p-Toluidinylnaphthalene-6-sulfonate (2,6-TNS) is a compound which is barely fluorescent in pure water but whose fluorescence can be strongly enhanced if the environment becomes hydrophobic, i.e. by the addition of suitable substrates such as proteins or 1, 4-alpha-D-glucans. The enhancement of fluorescence results from the formation of a 2,6-TNS/substrate complex. For linear and ramified 1, 4-alpha-D-glucans, the fluorescence intensities of the complexes depend linearly on their concentrations but nonlinearly on their average molecular weights (AMW). Thus, the fluorescence detector acts simultaneously as a linear detector concerning the concentration of 1,4-alpha-D-glucan and as a nonlinear mass-selective detector concerning its AMW. These properties have been used for the development of a fluorimetric 2,6-TNS-FIA methodology for the determination of beta-amylase activity, using amylose and amylopectin as substrates. The experimental data points, corresponding to the concentration of "detectable" substrate vs depolymerization time, were fitted using a two-parameter exponential decay curve, and the depolymerization rates at time zero were calculated. The depolymerization rates at time zero vs the corresponding initial substrate concentrations were fitted using the Michaelis-Menten hyperbola and the enzymic constants k(3) and K(m) for amylose (5.93 x 10(-3) g/microKat. min and 1.49 g/L, respectively) and for amylopectin (7.40 x 10(-3) g/microKat+. min and 1.65 g/L, respectively) were determined.
Batlle
N
N
Instituto de Agroquímica y Tecnología de Alimentos (CSIC), P.O. Box 73, 46100-Burjassot, Valencia, Spain.
Carbonell
J V
JV
Sendra
J M
JM
D000687:Amylopectin / Q000378:metabolism; D000688:Amylose / Q000378:metabolism; D017022:Flow Injection Analysis / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D005470:Fluorometry / Q000379:methods*; D007700:Kinetics; D009282:Naphthalenesulfonates / Q000032:analysis* / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D013379:Substrate Specificity; D001614:beta-Amylase / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D011108:Polymers; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D000688:Amylose; D000687:Amylopectin; D001614:beta-Amylase