Investigating enzyme action

• how do you calculate rate?

  1 / time
• what is the negative control reaction?

  set up to investigate using water (or boiled enzymes) in place of the functioning enzymes, used as a comparison to show that it is the enzyme action, rather than some other variables, which affects the reaction
• what are the two methods to measure reaction rate?

  • measuring rate that substrates are used
  • measure the rate products are formed
• what are examples of methods to measure reaction rate?

  • time how long colour change takes
  • measure a change in pH
  • use a colorimeter to measure the change in absorption at a particular wavelength of light
  • measure the volume and of gas produced or used up
• why control the temperature?

  • to ensure a constant temperature for enzymes
  • fluctuations in temperature will affect the rate of enzyme-controlled reaction
  • also, wait 5 mins before you start the timer to allow temperature to equilibrate
• why keep the SA:V ratio constant?

  the number of enzymes in contact the substrate molecule will affect the rate of reaction
• why use accurate measurements of enzyme solution volume?

  reaction rate depends on enzyme concentration - using an accurate measurement of volume keeps this constant
• why use accurate measurements of mass of living tissue?

  as you assume all the pieces of tissue contain same number of enzymes
• why use accurate measurements of volume or mass of substrate?

  changing the substrate concentration will change the rate of reaction
• why use pH buffers (solutions that maintain the pH at a set level by keeping the H+ concentration constant)?

  reaction rate depends on pH because of its effects on the tertiary structure and shape of the active site of the enzyme
• how is the initial rate of reaction calculated?

  by measuring the gradient of the steepest part of the curve
• why is it that the rate of reaction must be used?

  • only true point of comparison
  • only true maximum possible rate achieved
  • once reaction starts, the substrate is used up - therefore the only point where the substrate concentrations are equally (as a control variable they should have started this way) is the initial rate - this therefore is the only point that can be directly compared
• what is a negative control experiment for the method of the effect of pH on amylase using starch-agar plate?

  disc in boiled enzymes/amylase/or distilled water instead of functioning enzyme
• what are control variables in the effect of pH on amylase investigation?

  • total volume of enzyme and pH buffer is kept constant
  • keep enzymes concentration the same each time
  • ensure wells are same size
  • temperature kept constant at 35 degrees in dry oven
• what is the negative control experiment in the effect of temperature on catalase using potato cylinders?

  using glass discs (won‘t be denatured but mimic potato) - they take up the same volume as the potato disc
• what are the control variables in the effect of temperature on catalase investigation?

  • same type of potato (same natural concentration of enzyme)
  • same volume of pH 7 buffer & hydrogen peroxide
  • sand surface area of potato discs
  • concentration of hydrogen peroxide
  • constant air pressure
  • equal number of discs
• why should experiments be repeated?

  • makes it easier to identify anomalous data
  • averaging results by calculating mean assumes that wherever there are random uncertainties that cause inaccuracies, some of the repeats will be inaccurate below the real value and others will be inaccurate above the real value
  • an investigation with more repeats should give the most accurate results possible, therefore meaning the results are more reliable
• what is meant by serial dilution?

  • a repeated, stepwise dilution of a stock solution of known concentrations
  • the dilutions are often completed by factors of 10 to produce a range of concentrations
• why is it more accurate to perform a serial dilution, than to produce each concentration directly from the stock solution?

  to make the lowest concentration- you would either need to use a tiny volume of the stock solution, which couldn’t be measured accurately, or you will need to add it to a large volume of water that wouldn’t be feasible