Lab Exam Flashcards
What does the Beer-Lambert law state?
Light absorption is proportional to the number of molecules of absorbing substance through which the light passes
What is the Beer-Lambert equation?
A = Elc
If wavelength and path length are unchanged, how can absorbance be changed?
A change in concentration
Does absorbance have units?
NO
Does ONPG in solution absorb light?
Nope
What happens when ONPG is mixed with lactase?
Lactase catalyzes the hydrolysis of ONPG into ONP and galactose
How do you find the new concentration of a substance that is diluted in another substance?
Original [ ] / (final volume/original volume of what is diluted)
What is the value of Kw at 25 C?
10^-14 M^2
____ allows a buffer solution to maintain a near constant pH
The presence of both the weak acid and its conjugate base
What will the pH of a solution be if HA and A are in equal concentrations?
pH will equal pKa
Define buffer capacity
How much acid/base a buffer can be subjected to before the pH of the buffer changes dramatically
If 2 buffers have equal volumes and pHs, which will have a greater buffer capacity?
The one with a higher concentration
Why is Bradford assay popular?
It is very rapid and can detect very small quantities of protein
What is the Bradford assay based upon?
The binding of CBB to the protein under acidic conditions
What occurs when protein is added to the Bradford reagent?
The small amount of the free blue form can bind to the protein
What types of interactions occur in a Bradford assay?
- Ionic interactions with the side chains of basic residues
- Hydrophobic interactions with aromatic residues
Bradford assays can be monitored at ____ nm
595
What does a high Kf value mean?
A protein has a high affinity for its ligand
What is the Bradford assay graph equation?
Pt/Y = 1/nKf [1/1-Y] + Lt/n
How can you determine n?
Lt / y-intercept
How can you determine Kf?
1 / (n)(slope)
When are catalytic rate and substrate concentration almost proportional?
At low substrate concentration
What is Vmax?
The catalytic rate when the enzyme is saturated with substrate
What is the Michaelis-Menten rate equation?
Vo = Vmax[S] / Km + [S]
What is Km?
The substrate concentration that generates an initial rate equal to 1/2 of the maximum rate
Is Km dependent on enzyme concentration?
Nope
How are the y-values determined for a Lineweaver-Burk plot?
1/Vo
How are the x-values determined for a Lineweaver-Burk plot?
1/[S]
How is the slope determined for a Lineweaver-Burk plot?
Km/Vmax
How is the y-intercept determined for a Lineweaver-Burk plot?
1/Vmax
How is the x-intercept determined for a Lineweaver-Burk plot?
-1/Km
4-nitroaniline strongly absorbs light at ___ nm
410
What does beta-galactosidase do?
Catalyzes the hydrolysis of lactose into glucose and galactose
Why was ONPG used for the assays with E. coli and A. oryzae?
- ONPG is an artificial substrate of A. oryzae and E. coli
- Its chromophore (ONP) strongly absorbs light at 420 nm, while the natural substrate lactose doesn’t absorb light well
How is Kcat determined?
Vmax/Etotal
Irreversible inhibitors form ______ with an enzyme
Stable covalent bonds
Reversible inhibitors form ____ with an enzyme
Fleeting associations
Why are simple sugars easily detected?
Their anomeric carbons are free to reduce oxidizing agents
How can the amount of reducing sugars in a sample be quantified?
By measuring how much oxidizing agent is reduced
What happens to DNS when it is reduced?
It undergoes a colour change
How can you find concentration of reducing sugar?
Using the trendline equation, solve for x
How can you find total mass of reducing sugar?
Multiply the concentration of reducing sugar by supernatant volume and dilution factor
How can you find average reducing sugar %?
Mass of reducing sugar/sample mass * 100%
How can you find average % starch content?
Dry mass of starch/sample mass * 100%
What is transcription?
Conversion of DNA to RNA
What is translation?
Conversion of RNA to an amino acid sequence
What happens once DNA starts melting?
It becomes progressively easier to separate more of the DNA
What is Tm?
The midpoint of the transition between the double and single-stranded forms
DNA strongly absorbs light at ___ nm
260
Single-stranded DNA absorbs _____ more light than double-stranded DNA because ______
- 40%
- Decrease in base-base interactions
What is standard redox potential?
A measure of a species affinity for electrons
What does a high Eo value mean?
Higher affinity, so the compound can be more easily reduced
A substance with a ___ Eo will give electrons to a substance with a ____ Eo
Low, high
What is Eo proportional to?
The standard free energy of the reaction
How does the mitochondrial electron transport chain transfer electrons?
From NADH and FADH2 through a sequence of redox reactions to oxygen, which acts as the final electron acceptor
Does cytochrome c exist in an oxidized or reduced form?
Both (2+ or 3+)
What is Eo’?
The standard reduction potential at pH = 7.0
When is delta G = 0?
When the E value for one redox pair equals the E value for the other pair
How can you find fraction reduced?
A(sample) - A(fully oxidized) / A(fully reduced) - A (fully oxidized)
How can you find fraction oxidized?
1 - fraction reduced
What are the first steps of the calculations for making a buffer?
- Rearrange Henderson-Hasselbalch to solve for ratio of log [A-][HA]
- Plug in pH and pKa values
- Rearrange so [A] is by itself
- Plug value for [A] into [A] + [HA}
- Solve for [HA] then subtract that value from concentration of buffer to get [HA] and [A]
For a competitive inhibitor, what will change?
Km but not Vmax
For a non-competitive inhibitor, what will change?
Vmax but not Km
For a uncompetitive inhibitor, what will change?
Km and Vmax
