Biochemistry lab midterm

What is the first step to purifying an enzyme?

disrupt the cell

How can you disrupt a cell?

Hypotonic shock

Our wheat germ was placed in....

distilled water

Why was the wheat germ placed in distilled water?

its considered a hypotonic solution compared to the cell

When a wheat germ is place in distilled water, what happens to the cell?

it begins to swell and cause proteins to leak out

Name of our protein

acid phosphatase

What happens after the first centrifugation when disrupting the cell?

it separated ALL soluble protein from the insoluble parts of the cell

After the first centrifugation when disrupting the cell, the crude extract is the

supernant 1

Once we have the supernant 1, has any purification occurred?

NO

Why do we use cold water or keep everything on ice?

to inhibit proteases which degrade proteins

What are Proteases?

an enzyme that breaks down proteins into amino acids to be recycled in anabolic reactions making new proteins

What is p-Nitrophenyl Phosphate (pNPP)

a non-proteinaceous, non specific substrate used to assay proteins (alkaline and acid phosphatases)

What do we used to test for enzyme activity?

pNPP

What catalyzes the hydrolysis oof pNPP liberating inorganic phosphate and the conjugate base of PNP?

Acid Phosphatase

What stops the enzyme reaction and converts to its anion form giving it a yellow color?

Sodium Hydroxide or NaOH

True/False: we can monitor the activity of our enzymes of interest by observing the intensity of the color of the product para-nitrophenol (pNP)

True

What is the second step to purifying an enzyme?

separating wanted enzyme from unwanted enzymes

What is the balanced equation for Manganese Chloride?

MnCL2

What does MnCl2 do?

inhibit proteases and will cause some proteins to precipitate (but not acid phosphatase, our enzyme of interest)

Adding cold MnCl2 to Sup 1 and then centrifuging...

gets rid of some unwanted proteins and becomes Sup 2

After step 2, is there any purification done?

yes, a small amount of purification has been achieved

How to calculate 0.02 volumes of MnCL2 needed to add to your supernatant 1?

multiply 0.02 X your volume of Sup 1

The precipitate is also know as

the pellet

Adding 0.02 volumes of MnCl2 to your Sup 1 means

adding 2 ml of 1M MnCL per 100 ml of Sup 1

Example of calculating MnCl2 in your Sup: If your volume of Sup 1 is 27.5ml, how much MnCL would you need to add?

27.5 x 0.02 =0.55 ml or 550 μl

What is the 3rd step in purifying an enzyme?

Protein purification by precipitation

Adding COLD Ammonium Sulfate (NH4)SO2 is referred to as

salting out

-this is added until the solution is 33% saturated

Why do we add Ammonium Sulfate (NH4)SO2

it increases the ionic strength of the solution and the proteins become less soluble

Some unwanted proteins will precipitate (sink/fall out) from the solution at what concentration percentage?

33%

what does centrifugation do?

get rid of unwanted proteins

In step 3, what is happening in this suspension?

the NONPOLAR MOLECULES begin to coalesce and precipitate out the solution in a concentrated form

In step 3, is purification achieved?

some

Is our enzyme of interest still in the supernatant in Step 3?

yes, acid phosphatase remains

What is Step 4 in purifying an enzyme?

Separate wanted enzyme from unwanted protein

What are we first adding salt out the enzyme again?

cold ammonium sulfate until 60% saturated

At 60% concentration, what happens to the Acid Phosphatase?

it precipitates out

After we have our 60% saturated solution, we heat it to what temperature and for how long?

60 degrees C for 2 min

What happens when we heat the solution to 60 degrees C?

this causes other unwanted proteins to denature and precipitate

-the denatured proteins will not go back into suspension

Is the Acid Phosphatase (wAP) heat tolerant

yes, to 60 degrees C

After centrifuging Sup 4, what happens to the wAP?

it is in the pellet and should not be in the supernatant

What is step 6 in purifying an enzyme?

Separate wanted enzyme from unwanted proteins

What is used to resuspend the pellet from Sup 4?

Cold water

What does the cold water do to the ammonium sulfate?

It dilutes the ammonium sulfate

What enzyme is allowed to resuspend in solution by the dilution of ammonium sulfate?

Acid Phosphatase

Can denatured protein be re-suspended?

No

The resuspended pellet from Sup IV is centrifuged and allows

Acid Phosphatase to be separated from them (Sup V)

What is Step 7 of purifying an enzyme?

EDTA

What does EDTA do?

it inhibits proteases and they require metal ions to function and EDTA chelates the metal ions inhibiting the deviation of our enzyme

What is step 5 in purifying an enzyme?

separating wanted enzyme from unwanted proteins

What is it called when you add methanol?

solvent fractionation

What does adding menthanol do?

This separates proteins based on polarity. WaP will precipitate in this amount of methanol. Centrifugation will precipitate wAP.

What is Step 8 of Purifying an Enzyme?

Sodium Acetate (NaOAc)

Proteins only purify properly if?

they are folded properly in the correct combination of salt bridges, hydrogen bonds, and ionizable side chains

What makes the molecules less hydrophilic and less soluble in H2O?

Sodium Acetate (NaOAc) - Na++ neutralizes negative charge on phosphate groups

What precipitates nucleic acids within the dialysis tube?

NaOAc

NaOAc maintain a pH of what that is required by wAP to maintain its tertiary folds?

5

What is step 9 in purifying an enzyme?

Re-Suspend in cold water

Resuspending in cold water will

dilute out the methanol and allowing the Acid Phosphatase to re-suspend

Acid Phosphatase will be in the supernatant or precipitate once re-suspended in cold water?

Supernatant and should be relatively pure by now (Sup 6 and 7 are combined)

What does the dialysis do with the EDTA?

it will further dilute the methanol and ammonium sulfate as they will flow OUT of the bag

Which direction does the EDTA in regard to the dialysis bag?

it will flow INTO the bag until an equilibrium is reached

NaOAc will maintain a pH of

5

What was the solution makeup of the dialysis buffer?

500ml of an ice cold solution that has a concentration of 5mM EDTA and 100mM sodium acetate with a pH of 5.0

We want 500 mL of dialysis buffer @ 5mM EDTA and 100 mM NaOAc.

• we have 0.2 EDTA. We need to have 500ml solution of 5mM EDTA solution. How much EDTA do we need of 0.2 EDTA?

C1V1 = C2V2

C1= 0.2 M EDTA

V1 = unknown

C2= 5mM EDTA

V2= 500 mL

Convert everything to mM:

0.2 M EDTA = 200 mM EDTA

200 mM EDTA X V1 = 5 mM EDTA X 500 mL

V1 = 5 mM EDTA/200 mM EDTA X 500mL

V1 = 12.5 mL of 0.2 M EDTA per 500 mL

We want 500 mL of dialysis buffer @ 5mM EDTA and 100 mM NaOAc.

-We have 1 M NaOAc. We need to have a 500 mL solution of 100 mM NaOAc solution.

How much NaOAc do we need of 1 M NaOAc?

C1V1 = C2V2

C1= 1M NaOAc

V1 = unknown

C2= 100 mM NaOAc

V2= 500 mL

Convert everything to mM:

1M NaOAc = 100 mM NaOAc

1000 mM NaOAc X V1 = 100 mM NaOAc X 500 mL

V1 = 100 mM NaOAc /1000 mM NaOAc X 500mL

V1 = 50 mL of 1M NaOAc per 500 mL

how to prepare your dialysis buffer in a graduated cylinder

-50ml NaOAc

-12.5 ml EDTA