lab 3

Question 1

what factors affect enzyme activity?

Answer 1

temperature, pH, and substrate concentration, as well as the presence of other agents which may enhance or inhibit enzyme activity

Question 2

what does amylase do in this lab?

Answer 2

cleaves starch into maltose

Question 3

what does maltase do?

Answer 3

The enzyme maltase then converts maltose to glucose, which can be further broken down into carbon dioxide, water, and energy.

Question 4

what do we add to the solution in order to detect the presence of starch?

Answer 4

iodine

Question 5

what colour does a solution containing starch turn when iodine is added?

Answer 5

blue/black. if only a little bit, yellow/bown

Question 6

what is the control in this lab, used to set the spectrophotomer? Why do we do this?

Answer 6

100% transmittance using an iodine control, then a 100% transmittance reading should be reached upon complete hydrolysis of starch in the experiment

Question 7

what is the goal of this lab?

Answer 7

it will be possible to determine the optimal pH or the optimal enzyme concentration for starch hydrolysis

Question 8

what is a valid rep of enzyme activity?

Answer 8

An increase in % transmission over time reflects the rate of substrate (starch) disappearance

Question 9

how do we calculate the rate of reaction for starch

Answer 9

A rate is expressed as “units/time”. In this case, the rate of the enzyme reaction will be expressed as “mg starch hydrolysed/minute”. To calculate the rate, we will simply determine the time it took to hydrolyse half of the starch present at t = 0. To do this, we will draw a horizontal line at 50% transmission and follow down where the transmission curve intersects this line to find the time taken. To express the rate as mg starch/minute, we must know how much starch has been hydrolysed at the 50% point, and this is calculated as initial mg starch x 0.5. For example, under a given pH condition, 25 ml of starch at a stock concentration of 8 mg starch/ml means that 200 mg of starch was present in the flask at t = 0. 50% transmission occurs at 10 minutes meaning that 100 mg was hydrolysed in 10 minutes, therefore the rate is 10 mg starch hydrolysed/minute.

Question 10

how do we determine the rate of reaction if no hydrolysis occurs or if the transmission value does not reach 50 percent?

Answer 10

Note that if no hydrolysis occurs within the 25 minutes, the rate should be considered as 0, and if the transmission values do not reach 50%, a rate could be calculated from a lower transmission value, such as 20%, by performing the same calculations to obtain mg starch hydrolysed (eg. initial mg starch x 0.2).

Question 11

how do we find the rate of reaction vs ph or enzyme concentration graph?

Answer 11

for each one of our points, we find the rate of reaction and then we plug each concentration/ph as x and the rate as y

Question 12

Why is the optimal PH for this reaction?

Answer 12

5

Question 13

What is the optimal enzyme concentration for this reaction?

Answer 13

0.3mg/ml , because beyond this point, the function has hit its plateau and the enzyme is in excess

Question 14

In general, how does temperature affect enzyme activity, ie. explain why there is a peak rate and why activity is slower at more extreme temperatures.

Answer 14

Most enzymes have an optimal temperature at which the rate of reaction is fastest. Enzyme reactions occur slowly or not at all at low temperatures. The rates increase (within limits) because substrates collide with the active sites more frequently as temperature increases. Higher temperatures rapidly inactivate most enzymes. The molecular conformation of the protein becomes altered as the thermal agitation of the enzymes molecule disrupts H bonds, ionic bonds, and other weak interactions responsible for its 2o, 3o and 4o structures and the protein denatures

Question 15

How does pH affect enzyme activity?

Answer 15

Most enzymes have an optimal pH and are active only over a narrow pH range. Changes in pH alter charges on the enzyme which in turn affect ionic bonds that contribute to 3o and 4o structures, thus changing the protein’s conformation and activity.

Question 16

Why does the enzyme activity plateau (level off) with increasing enzyme concentrations?

Answer 16

If pH, temperature and [S] are constant (with [S] initially in excess) then the [enzyme] is the rate limiting factor and the initial rate of reaction will be directly proportional to the concentration of enzyme present. However because [S] is constant, the reaction rate will eventually slow regardless of [E] because less substrate is available. In other words, at high [E], substrate becomes limiting.

Question 17

Why does increasing the substrate concentration increase the rate of the reaction?

Answer 17

If the [E] is constant then the initial rate of reaction will be proportional to the substrate present, i.e., the [S] is the rate limiting factor at lower concentrations so the rate of reaction will be directly proportional to [S]. However, note that at higher [S] the enzyme molecules become saturated with substrate and then increasing [S] does not increase the rate any further.

Question 18

what is 100 percent transmittance?

Answer 18

no starch present

Question 19

how do we freeze the reaction?

Answer 19

cover with parafilm and invert tube

Question 20

What is the use of this graph? What does it signify?

Answer 20

This graph allows us to check the reaction rates at different pHs. We will have a similar graph for enzyme concentration. The title would be: amylase activity at different PH: % transmittance as a function of time