Bio1022 Prac 3 Metabolism Barley
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You have performed the experiment (your practical 3, Metabolism experiment). You wish to tell your friend about this experiment so they can replicate it exactly. Write a methods section for this practical.
This methods section should be brief. Do not write lists of materials and do not write in bullet-points or numbered form – write full sentences. You must describe how the activity of amylase was calculated in the germinating barley (section 2g). Describe the steps involved using words, not calculations. The methods should also explain (i) why a boiled control was used in 2f, and (ii) how to correct the value of amylase activity for any of the stages if an achromic point had been reached for the control experiment.
(Recommended word length 250-350 words).
Weigh 10 seeds of germinating barley and then crush them for about 3 minutes, adding 10 mL of buffer gradually. Filter the amylase extract to remove solid residuals and record the volume of extract obtained. Transfer 5 mL of the amylase paste to a measuring cylinder and add 20 mL of buffer obtaining a total volume of 25 mL hence diluting the amylase extract by a factor of 5.
To prepare a control extract, transfer 5mL of the diluted amylase extract to a test tube and place it in a boiling water bath for 10 minutes. Boiling the control extract denatures the amylase enzyme that is expected to act on starch. This is done to the boiled control to ensure that in the absence of the amylase enzyme, an inorganic component is not responsible for the reaction of starch to maltose. While the control test tube boils for 10 minutes, prepare the ceramic plates by adding one drop of iodine into each of the 21 wells of the ceramic plates.
To prepare the reaction mixture, add 5mL of buffer to 1mL of the 0.5% starch solution in a test tube. Place a drop of this mixture to the test well of the ceramic plate and observe the dark blue control colour of the starch and iodine mixture. Add 1mL of diluted amylase extract to the test well and the first well of ceramic plate and continue this addition every minute moving along the ceramic plate’s wells until the point where there is no longer a colour change, also called the achromic point. Repeat the same procedure for the 10 dormant seeds, the 10 whole barley seedlings and use the original boiled control extract for all experiments.
Calculate the amylase activity by calculating the concentration of starch added to the reaction tube and then calculating the hydrolysed starch concentration to reach the achromic point per minute. Next, calculate the concentration of barley tissue in the amylase extract in g/mL. Finally, calculate the amylase activity by dividing the concentration of hydrolysed starch and the concentration of barley tissue in the extract. The value obtained from this division must be multiplied by 5. If a reaction occurred in the control experiment then the activity measured in the control experiment would need to be subtracted from the experimental reaction rate calculations.
To determine and identify maltose as the product obtained from starch hydrolysis, place 2 mL of the amylase reaction mixture to a test tube with 2 mL of Benedict’s reagent. Prepare a control by mixing 5 mL of buffer and 1 mL of 0.5% starch solution in a clean test tube. Transfer 2 mL of this mixture to a test tube filled with 2 mL of Benedict’s reagent and examine the presence of cuprous oxide precipitate which is found when boiled with maltose.
Your friend has performed the experiment and collected the data below. From this data, calculate the amylase activity of the