Maltase Assay

Method: The enzymatic activity is determined by measuring the increase in absorbance at 400 nm caused by the hydrolysis of p-nitrophenyl-α-D-glucopyranoside. One unit hydrolyzes one umole of p-nitrophenyl-D-glucopyranoside (PNPG) at 37°C, pH 6.8, under the specified conditions.

Reagents

• 0.067 M Potassium phosphate, pH 6.8 with 0.001 M dithio-threitol (DTT).
• 0.01 M p-nitrophenyl-α-D-glucopyranoside (PNPG). Substrate may be aliquoted into vials and frozen for future use. Once thawed, the substrate may not be re-frozen.

Enzyme

Dilute immediately before use in 0.067 M Potassium phosphate to obtain a rate of 0.02-0.04 ΔA/minute. The protein may be determined as follows:

Procedure

Adjust spectrophotometer to 400 nm and 37°C and pipette into cuvettes as follows:

	Test	Blank
Phosphate buffer	2.8 ml	2.9 ml
PNPG substrate	0.1 ml	0.1 ml

Incubate in spectrophotometer for 5-7 minutes to achieve temperature equilibrium and establish blank rate, if any. Add 0.1 ml diluted enzyme to test cuvette and mix. Record increase in A₄₀₀ for 5-6 minutes. Calculate ΔA₄₀₀/minute from the initial linear portion of the curve.

Calculation