Peroxidase Enzyme Activity Lab

Flashcards covering the peroxidase enzyme laboratory practical, including spectrophotometric assay principles, guaiacol oxidation, effects of temperature, pH, and hydroxylamine inhibition.

What is the primary biological function of the enzyme peroxidase in cells?

To convert toxic hydrogen peroxide ($H_2O_2$), produced during certain metabolic reactions, into water and another harmless compound.

How is the structure of peroxidase characterized in terms of its size?

It is a large protein composed of over 300 amino acids.

What general reaction equation describes the activity of peroxidase using an electron donor ($RH_2$)?

$$H_2O_2 + RH_2 \rightarrow 2H_2O + R$$

Which dye serves as the electron donor (reductant) in this lab assay, and what visible change occurs when it is oxidized?

Guaiacol is used; it is colourless in its reduced state and turns brown when oxidized.

What is the specific reaction sequence for the peroxidase-catalyzed oxidation of guaiacol?

$$H_2O_2 + \text{guaiacol (colourless)} \xrightarrow{\text{Peroxidase}} 2H_2O + \text{oxidized guaiacol (brown)}$$

At what wavelength does oxidised guaiacol absorb light in the spectrophotometer?

$$500\,\text{nm}$$

Which four temperatures are tested in Experiment 5.1 to investigate the effect of temperature on peroxidase activity?

In an ice bath at $0\text{--}4\,^\circ\text{C}$, at $25\,^\circ\text{C}$, at $37\,^\circ\text{C}$, and at $65\,^\circ\text{C}$.

What are the pH values of the buffers supplied to examine the effect of pH on peroxidase activity in Experiment 5.2?

pH 3, pH 5, pH 7, and pH 9.

How long must the test tube pairs be placed in the water baths to reach their target temperature before mixing?

$$20\,\text{minutes}$$

Why must the timer be started immediately upon mixing the tube contents rather than after placing the cuvette in the spectrophotometer?

The reaction begins as soon as the tubes are mixed; starting the timer later causes data lines not to pass through the origin ($0\,\text{time}$ and $0\,\text{absorbance}$ relative to the blank).

At what intervals and for what total duration are spectrophotometer readings recorded at $500\,\text{nm}$?

Every $20\,\text{seconds}$ for a total duration of $3\,\text{minutes}$ ($180\,\text{seconds}$).

What is the composition and purpose of Tube 1 in the mixing protocols?

It acts as the blank to adjust the spectrophotometer, containing $5\,\text{ml}$ buffer, $2\,\text{ml}$ $H_2O_2$, and $1\,\text{ml}$ guaiacol (with no peroxidase enzyme added).

Which molecule acts as a competitive inhibitor in Experiment 5.3, and why does it inhibit peroxidase activity?

Hydroxylamine ($HONH_2$); its molecular structure is similar to hydrogen peroxide ($HOOH$), allowing it to bind to the active site and inhibit enzyme activity.

What pretreatment procedure is required for peroxidase before setting up Experiment 5.3?

$2\,\text{ml}$ of peroxidase solution must be pretreated (incubated) with either $1\,\text{ml}$ of $2\%$ hydroxylamine or $1\,\text{ml}$ of pH 7 buffer at $25\,^\circ\text{C}$ for $15\,\text{minutes}$.

What were determined to be the optimal temperature and optimal pH for peroxidase activity in this laboratory practical?

The optimal temperature was $37\,^\circ\text{C}$ and the optimal pH was 5.

What effect does heating peroxidase at $100\,^\circ\text{C}$ for $20\,\text{minutes}$ have on its activity, and what causes this effect?

It stops the enzyme from breaking down hydrogen peroxide because the high temperature irreversibly denatures the protein and destroys its active site shape.

Which graphical feature of the experimental data lines indicates the reaction rate of peroxidase?

The slope of the line of best fit.

What physiological example illustrates enzymes with different pH optima in the human digestive system?

Pepsin operates in the stomach at an optimum below pH 3; upon entering the small intestine, pancreatic and liver bicarbonate raises the pH above 7, stopping pepsin activity while other enzymes take over digestion.