Measuring Enzyme Activity

• Collision Theory: For a reaction to occur, enzyme and substrate molecules must collide with sufficient energy and in the correct orientation. Measuring activity quantifies the frequency of successful collisions.

• Substrate Limiting Factor: As a reaction progresses, the concentration of substrate decreases. This leads to fewer collisions per second, causing the reaction rate to naturally decline over time.

• Product Inhibition: In some cases, the accumulation of product molecules can interfere with the enzyme's active site, further slowing the measured rate as the reaction continues.

1. Gas Collection

• This method is used when a reaction produces a gaseous product (e.g., Oxygen from Hydrogen Peroxide breakdown). The gas is collected in a gas syringe or an inverted measuring cylinder over water to measure volume over time.

2. Colorimetry

• Colorimetry measures the intensity of color in a solution. A colorimeter passes light through a sample and measures Absorbance (how much light is blocked) or Transmission (how much light passes through).

• It is highly effective for reactions involving a color change, such as the disappearance of blue-black starch when treated with amylase in the presence of iodine.

3. Mass Loss

• If a gas is produced and allowed to escape, the total mass of the reaction vessel will decrease. Placing the reaction on a high-precision digital balance allows for real-time tracking of the reaction rate.

• Absorbance vs. Transmission: In colorimetry, as a colored substrate is broken down, the Absorbance decreases while the Transmission increases. It is vital to choose the correct filter (complementary color) to maximize sensitivity.

• Calculating the Gradient: To find the initial rate from a graph, always draw a tangent to the curve at $t = 0$. The gradient of this tangent ($m = \frac{\Delta y}{\Delta x}$) gives the initial rate.

• Control Variables: When investigating the effect of one factor (e.g., temperature), you MUST explicitly state that other factors like pH, enzyme concentration, and substrate concentration are kept constant to ensure a fair test.

• Calibration: Always mention 'zeroing' or calibrating a colorimeter using a 'blank' (usually distilled water or a starting reaction mixture) to ensure all subsequent readings are relative to a standard.

• Ignoring the Initial Rate: Students often calculate the average rate over a long period. This is inaccurate because the rate changes as substrate is consumed. Always prioritize the initial rate.

• Equating Activity with Concentration: High enzyme activity does not always mean high enzyme concentration; it could be due to optimal temperature or pH conditions.

• Temperature Fluctuations: Enzymes are highly sensitive to heat. Failing to use a thermostatically controlled water bath is a frequent source of experimental error.