Properties of Enzymes (L.P 2)

Question 1

What were the 3 Independent Variables?

Answer 1

• Enzyme concentration
• Initial substrate concentration
• pH

Question 2

What was the dependent variable?

Answer 2

-Rate of reaction

Question 3

What enzyme did we use in the experiment?

Answer 3

-Alkaline Phosphatase

Question 4

What does Alkaline Phosphatase do?

Answer 4

• It removes phosphate from molecules within the cell

- This leads to the product of Para-nitrophenol (pNP) which appears as yellow

Question 5

What is the relationship between the concentration of pNP in a solution and absorbency?

Answer 5

-The greater the concentration of the pNP the more blue light it absorbs & the higher its absorbance

Question 6

How can we measure the rate of reaction?

Answer 6

• How fast the yellow color appears (pNP)

- How fast the enzyme works

Question 7

How many syringes did we use & what were they?

Answer 7

• 11 different syringes,
• 6 for substrate concentration
• 5 for enzyme concentration
• 1 for the buffer

Question 8

How do we calibrate the spec 20?

Answer 8

1) Turn it on and make sure it is on infinity by turning the left knob
2) insert the blank tube & adjust so that it reads 0 absorbance by using the right knob
3) then make sure its zero on the read-out graph on the computer

Question 9

How do you make a blank test tube?

Answer 9

1) Add 1 mL of distilled water
2) add 1.6mM of pNPP
3) add 1mL of buffer 10.0

Question 10

What is the procedure for the Enzyme Concentration run?

Answer 10

1) add 1mL of buffer 10.0
2) add 1.0 mL of 1.6 mM pNPP
3) add enzyme really quickly & put the parafilm over the mouth of the test tube to shake it
4) put the tube into the spec 20

Question 11

What times did we snap the Blue and Red lines to?

Answer 11

at time 2 and 62

Question 12

How do we calculate the change of absorbance for the 60secs?

Answer 12

do red cursor minus blue cursor

Question 13

What was the result of the dependence of Enzyme Concentration on Change of Absorbancy?

Answer 13

-It was a linear relationship because the more enzyme we have the linear the relationship

Question 14

What was the procedure for the Substrate Concentration run?

Answer 14

1) add 1.0mL of buffer and pNPP to a test tube

2) add 1.0 mL of enzyme 8 really quickly

Question 15

What was the result of the dependence of Substrate Concentration on Change of Absorbancy?

Answer 15

• The graph was kinda like a hookshot w/ a limit at the top= Saturation
• Since all enzyme molecules are working, the substrate molecules must wait bc the enzymes are already going as fast as they can

Question 16

What was the procedure for the Effect of pH run?

Answer 16

• Same as substrate concentration test

- Except we’re also using pH 8.5 and 11.9 in addition to the 10.0

Question 17

What was the result of the dependence of pH on the Change of Absorbancy?

Answer 17

• It was an upside-down parabola
• This is bc pH changes the shape of the enzyme causing it to unfold or denature depending on which extreme its on
• The optimal pH for the reaction was 10.0 (buffer)