Method: The determination of reaction velocity is based upon the method of Folk and Schirmer (1963). The rate of hydrolysis of hippuryl-L-phenylalanine is determined by measuring the increase in absorbance at 254 nm. One unit hydrolyzes one micromole of hippuryl-L-phenylalanine per minute at pH 7.5 and 25°C under the specified conditions.

Reagents

• 0.025 M Tris⋅HCl buffer containing 0.5 M sodium chloride, pH 7.5
• 0.001 M Hippuryl-L-phenylalanine in 0.025 M Tris⋅HCl, pH 7.5 with 0.5 M sodium chloride
• 10% Lithium chloride

Enzyme

Dissolve in 10% lithium chloride to a concentration of 1-3 units per ml. The enzyme crystals are not readily soluble in the diluent. Do not use solution for assay until the solution has cleared.

Read A₂₇₈ in cold 10% lithium chloride.

Procedure

Adjust spectrophotometer to 254 nm and 25°C.

Pipette 2.0 ml of substrate into each cuvette and incubate in spectrophotometer at 25°C for 3-4 minutes to reach temperature equilibration and establish blank rate, if any. Add 0.1 ml of diluted enzyme and record increase in A₂₅₄ for 3-5 minutes. Determine ΔA₂₅₄ / minute from the initial linear portion of the curve.

Calculation