Bio 173 Exam 1

Question 1

Hypothesis

Answer 1

a testable prediction based on observations and assumptions
must be falsifiable

Question 2

Prediction

Answer 2

a description of an expected outcome in the test (experimental) group and in each control group – Confirming a prediction supports (but does not “prove”) that hypothesis – Negating a prediction suggests that it may be wrong

Question 3

positive control

Answer 3

response expected, ensures that the setup works properly and provides a baseline (antibiotics)

Question 4

negative control

Answer 4

no response expected, ensures results are due to the variable being tested and not something else (placebos)

Question 5

enzyme

Answer 5

catalytic protein (think lock and key/puzzle piece)

Question 6

How do enzymes lower the activation energy barrier?

Answer 6

Lowering the activation energy (alternate pathway)

Question 7

What is an exergonic reaction?

Answer 7

A spontaneous reaction, happens naturally

Question 8

factors that influence catalysis

Answer 8

temperature
pH
substrate concentration
enzyme concentration
activator presence
inhibition presence

Question 9

What reaction does polyphenoloxidase catalyze?

Answer 9

catechol to orthoquinone

Question 10

how to measure reaction rate

Answer 10

slope
initial reaction rate is the first 2 min slope

Question 11

how does enzyme conc affect the reaction rate

Answer 11

Determines how fast the rate of the reaction will be

Low concentration - fewer available active sites, reaction is limited by the number of enzymes

High concentration - reaction is faster because enzymes can find substrates to bind to quicker

Question 12

Vmax

Answer 12

where the reaction levels off on the chart
maximum rate of reaction when all the enzyme have been used up, adding substrate wont do anything past this point

Question 13

Km

Answer 13

[substrate] when the reaction is at halfway

Question 14

cofactor role

Answer 14

required for the reaction to occur, it primes/shapes the activation site

Question 15

competitive inhibitor

Answer 15

goes into the enzyme - increases Km but not vmax - % inhibition varies at different concentrations

Question 16

non-competitive inhibitor

Answer 16

goes into an alternate area (allosteric site) and changes the overall shape - decreases vmax but not Km - % inhibitor is constant for all concentrations of substrates

Question 17

how is Vmax affected by inhibitors

Answer 17

noncompetitive inhibitors - decreases Vmax
competitive - none

Question 18

calculate % inhibition

Answer 18

(Rate without inhibitor - rate with inhibitor)/(rate without inhibitor) x 100%

Question 19

what does % inhibition means

Answer 19

how much of enzyme is used up

Question 20

Effect of running the reaction at extreme temperatures (very hot, very cold)

Answer 20

45 C fastest rate, 20 C (control), 0, 65
At 45, the molecules are excited - more collisions make it more likely for the reaction to occur, making it happen faster (shift the chart left)
At 0 molecules have a lot less kinetic energy, very slow
At 65, the enzyme is denatured (there is low to no reaction)

Question 21

Effect of freezing and thawing the enzyme vs boiling it and letting it cool

Answer 21

Control - never frozen or boiled
Fastest reaction is cold tied with the control, then boiled (denatured)

Question 22

Is Potassium arsenite an inhibitor

Answer 22

6mm catecol with inhibitor
6mm catecol without inhibitor
24 mm catecol with inhibitor
24 mm catecol without inhibitor
If we vary the substrate concentration and its a competitive inhibitor, we expect less inhibition
Less substrate to compete with inhibitor for active site
If its a noncompetitive inhibitor, % inhibition is the same
It is barely an inhibitor for PPO, % inhibitor was ~5 ish on each
No sulphohydride groups in polyphenoloxidase

Question 23

Does polyphenoloxidase have a metal cofactor?

Answer 23

Cofactor - required for the reaction to occur
primes/shapes active site
Regulates things in the body
Blood - iron is a cofactor for hemoglobin
Isolate the cofactor (if it is even there)
Add a keylator (substance that pulls it out)
PTU is copper only
Potassium cyanide is copper and iron
Control, ptu and potassium cyanide
PTU and potassium cyanide both had slowed reactions, meaning that copper is the cofactor of pilyphenoloxidase

Question 24

The effect of increasing salt

Answer 24

Ions in salt solutions can destabilize ionic bonds but its not an inhibitor
It is a universal inhibitor but not specific to an enzyme, it does not change the shape or go into the enzyme to prevent the substrate
Salt is denaturing the enzyme
Needs to be pretreated (denaturation doesn’t happen immediately)
Run with increasing salt concentration
Control, high concentration, low concentration
As salt increases, reaction rate decreases (to a point)
Biologically relevant because of saltwater ecosystems have very specific concentrations
Ice caps are melting, diluting the salt concentration, impacting these systems

Question 25

What is in a PCR cocktail (master mix)? What is the function of each?

Answer 25

Taq polymerase - heat stable DNA polymerase, binds to 3’ end of primer
Pair of primers -DNA/RNA used to start synthesis
Very specific sequence
Short fragments (18-25 nucleotides)
Nucleotides - each type (dATP, dTTP, dGTP, dCTP)
Buffer - keeps pH constant to prevent denaturation
Source of DNA (freezer stock/colony) to repair
Don’t need ligase because you are melting everything down

Question 26

What is the purpose of positive and negative controls in PCR?

Answer 26

Amplify (make more) of a gene of interest so you can study it
Makes it possible to detect if a gene is present or not
Run on gel electrophoresis to see if it is present
Used as a template for DNA sequencing - find the base pairs of each part

Question 27

Three steps of PCR

Answer 27

Denaturation (very hot)
Annealing (slightly cooler)
So the primers can bind
Expansion (much cooler)
So DNA polymerase can act, bind and create new DNA

Question 28

Why use freezer stocks

Answer 28

keep colony alive and stable