Dextranase Assay

Method: A modification of that described by Janson and Porath (1966). One Unit of activity causes the release of one micromole isomaltose from dextran per minute at 37°C and pH 6.0 under the specified conditions.

Reagents

• 0.1 M Potassium phosphate, pH 6.0
• 2% Dextran. Dissolve 1.0 gm dextran 500 in 30 ml 0.1 M potassium phosphate pH 6.0. under the specified conditions. Bring to a final volume of 50 ml.
• 2% Sodium hydroxide
• Dinitrosalicylic acid color reagent. Prepare by dissolving 1.0 gm of 3,5-dinitrosalicylic acid, 200 mg phenol, 50 mg sodium sulfite, 20 gm sodium potassium tartrate tetrahydrate in 60 ml of 2% NaOH. Q.S. to 100 ml with 2% NaOH. Protect from carbon dioxide and store no longer than 2 weeks.

Enzyme

Dissolve enzyme at one mg/ml in reagent grade water. Immediately prior to use, dilute further to 5-20 micrograms/ml.

Procedure

Into a series of numbered tubes pipette 1.9 ml dextran substrate. Include one tube to be used as a blank. Incubate in a 37°C water bath. At timed intervals, to each tube add 0.1 ml of an enzyme dilution. To the blank, add 0.1 ml of reagent grade water in place of the enzyme.

Incubate at 37°C for 30 minutes. Stop the reaction at timed intervals by removing one ml aliquots of enzyme-substrate mix to tubes containing one ml dinitrosalicylic acid reagent. Incubate for 15 minutes in a boiling water bath. Cool to room temperature, add 10 ml reagent grade water to each tube, mix and read A₅₄₀.

Calculation

Determine micromoles of maltose in aliquot from a standard curve.