**Experiment 1** Students investigated the rate of enzymatic hydrolysis of o-nitrophenyl-\… — ACT

Experiment 1

Students investigated the rate of enzymatic hydrolysis of $o$ -nitrophenyl- $\beta$ -D-galactopyranoside ( $ONPG$ ) catalyzed by the enzyme lactase. The reaction yields $o$ -nitrophenol, a product that absorbs light strongly at a wavelength of $420\text{ nm}$ . To accurately measure the rate of product formation using a spectrophotometer without error from background absorbance or premature reaction, which of the following represents the correct chronological sequence of procedural steps the students must perform?

1Calibrate the spectrophotometer to zero absorbance ( $100\%$ transmittance) using a 'blank' cuvette containing only buffer solution.
2Pipette the buffer solution and substrate ( $ONPG$ ) solution into a clean reaction cuvette and allow it to equilibrate to the target temperature.
3Add the lactase enzyme solution to the reaction cuvette, mix the contents quickly by inversion, and start the timer immediately.
4Insert the reaction cuvette into the spectrophotometer sample compartment and close the light-tight lid.
5Record the absorbance values displayed on the spectrophotometer at $30\text{-second}$ intervals over a total period of $3\text{ minutes}$ .

Answer

The correct procedural order is: (1) Calibrate the spectrophotometer with a blank cuvette containing buffer solution, (2) Combine substrate and buffer in the reaction cuvette and temperature-equilibrate, (3) Add lactase enzyme solution, mix quickly, and start the timer, (4) Insert the reaction cuvette into the spectrophotometer sample holder and close the lid, (5) Record absorbance values every 30 seconds for 3 minutes.

The proper scientific workflow begins with instrument baseline calibration using a blank cuvette containing only buffer to zero out background absorbance. Next, the reaction vessel is prepared by combining buffer and substrate (

ONPG

) to reach the desired temperature. Adding the enzyme initiates the catalytic reaction, which requires immediate mixing and starting the timer (

t = 0\text{ s}

). The cuvette is then swiftly transferred into the instrument's light-tight chamber to prevent ambient light distortion, after which kinetic absorbance readings are systematically logged every 30 seconds.

Step-by-Step Solution

Identify baseline calibration requirements.

Spectrophotometer zeroing must precede any measurement.

Calibrating with a blank removes baseline optical absorption by solvent and cuvette walls.

Determine reaction mixture preparation steps prior to initiation.

Substrate and buffer are combined and thermal equilibrium established.

Enzyme activity is temperature-dependent, so temperature must be stabilized before reaction initiation.

Pinpoint the exact initiation point of the reaction.

Enzyme introduction defines

t = 0\text{ s}

The reaction begins immediately upon enzyme addition, necessitating rapid mixing and timer start.

Determine the physical placement of apparatus components.

Cuvette is transferred into the sample compartment and closed.

Closing the lid prevents ambient room light from interfering with detector readings during measurement.

Establish data collection timeline.

Absorbance readings are documented periodically at

30\text{-second}

intervals.

Periodic sampling allows calculation of the initial reaction velocity.

Key Concept

Sequencing Spectrophotometric and Enzymatic Assay Procedures

Estimated Time:1m 30s

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Step-by-Step Solution

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