Differential Scanning Calorimety

Question 1

What is differential scanning calorimetry

Answer 1

Measures differences in heat capacity between the sample and reference due to phase changes

This phase change could be due to denaturation of the protien

Question 2

How does DSC actually measure things

Answer 2

It measures the heat that needs to be added to change the temp of the sample

Does this by adding two amounts of heat,

To the sample (qs) and to the reference (qb) through electrical energy to slowly raise the temp of the cells at a constant rate

Question 3

Explain the actual machine of the DSC

Answer 3

You have the reference cell and the sample cell

Sample cell has the bio molecules and buffer

Reference has buffer

The two cells are insulated in a thermal jacket and are warmer than the outside, Which is why there is small amount of heat loss from the cells

Question 4

In DSC how is the temperature difference actually made

Answer 4

The samples has a higher heat capacity (can absorb more heat) during the phase change so you can see the difference in temp between the sample and the reference

Question 5

In DSC, what equalizes the temp when the temperature difference is made

Answer 5

A feedback loop that monitors the change in temp and adds more heat (Q) to make the temp equilibrium

Question 6

What does delta Q = qs - qb mean

Answer 6

The difference between the heat given to the sample and to the reference (blank)

Question 7

What is special about the pressure in DSC

Answer 7

Since the liquid in the cell can expand, The process is happening at constant pressure

This means no work is being done

so delta Q = delta H and the Cp = delta Q/ delta T

Question 8

What is a thermogram

How do we get info from it

Answer 8

This is the result from the DSC

It’s a plot of Cp vs temp

The temperature at the peak of the plot is the melting temp (50% folded and 50% unfolded)

The area under the peak is delta H

Question 9

Explain the calculation for measuring delta H per gram in DSC

Answer 9

Find area : 1/2 b x h = ____ micro joules

Find mg of protien : mg/mL x mL = __mg

Delta H = area / mgx10^-3 = J/g

MEASURE BASE AT EXACTLY WHERE THE PEAK STARTS AND ENDS (SMALLER THAN YOU THINK)

Question 10

Explain the calculation for measuring delta S per gram in DSC

Answer 10

Delta S = delta H / T

Find peak temp (melting temp):
degree + 273 K = __K

Use delta H you found

Delta S = Delta H/ T in K = ____ J/ g K

Question 11

What are the main parameters for DSC

Answer 11

the starting and final temp: want to start below the melting temp and end well above

Scan rate has to be slow: not a fast experiment

Want to scan multiple times: meaning you do the experiment once then decrease the temp to do it again, but the unfolding has to be reversible to do this

The pre and post thermostat: how long you maintain the temp before the experiment , 15 min to make sure temp at equilibrium

What temp you keep it at after the experiment, 10 degrees to keep the instrument good

Question 12

What are the type of lipid vesicles and their characteristic

Answer 12

DMPC: mammalian lipid, rigid gel phase, zwitterionic

DMPE: bacterial lipid, h bonded, tight packed

DMPG: bacterial lipid, negatively charged

Question 13

Explain how the DSC of lipid vesicles changes based on the binding of peptides to them

Answer 13

Binding of the peptide to the lipid vesicles increases its melting temperature, more stable

Shifts to right

Question 14

Explain lipid pre transition

Answer 14

At lower temp the lipid is in the gel phase and ordered

pre transition: is before the main transition where the lipid becomes less ordered, displaced and is in the ripple phase

Main transition: At the higher temp the lipid is in the fluid phase

Question 15

How can DSC be used to judge the quality of protiens

Answer 15

In comparing three batches of the same protien their melting temps should be the same

But DSC can show they they are not meaning the quality of the protiens are bad since they have differences

Question 16

How can DSC be used to see increasing stability of protiens

Answer 16

In more stable it would have a higher melting temp

So if trying to make a drug do DSC on diff forms of that drug then see which has the higher melting temp to see which is most stable

Question 17

Adavantage of DSC

Answer 17

Most accurate way to measure thermodynamic parameters

Universally applicable: doesn’t need a spectroscopic handle (like a label or covalently attached probe)

Non destructive, so reversible (in the case of lipids that’s true but not so true for protiens)

Question 18

Disadvantage of DSC

Answer 18

Has low sensitivity (needs high concentrations)

Slow speed

To get good values and have many runs, phase change needs to be reversible and has to be a well behaved system

Question 19

Explain what the p53 case study shows

Answer 19

P53 binds to double stranded dna

When bound, the melting temp is higher than just p53

When the p53 and dna dissociate, the p53 unfolds immeadiately and so does the dsDNA

Can see the binding or dissociates of p53 and DNA in the plot, but actually measuring them is difficult

Question 20

If you have measurements in DSC and the protien doesn’t unfold reversibely what does this mean

Answer 20

The data is qualitative not quantitative