The Road to Calorimeter Master Vol.8
Measuring RNase A with DSC! Part 2

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The characters and story presented here are fiction.
For technical content, we have received advice from Professor Fukada, a guest researcher at Osaka Prefecture University.

From the “Download” button at the bottom of the page, you can download the “VP-DSC Analysis Procedure” mentioned in the text.

(Continued from Part 1

The baseline of the control measurement is stable. Remove the pressure cap at around 30°C, and fill the sample. Use the ThermoVac to degas the sample with the Timer. Once degassing is complete, remove the pressure cap and set the pressure plug on the pressure sensor. This time, without using a filling funnel, carefully insert a needle connected to a lure cap syringe into the cell without touching the bottom, and remove the buffer from the sample cell. The degassed sample is now filled. After 15 minutes of PreScan Thermostat, it’s time for Final Equilibration. The DP value is approaching zero, so all is good!

Mr. Nakamura, is everything going smoothly?

	Yes, the DP value seems fine. Now we just wait for the measurement results.

By the way, did you change the Number of Scans?

	Oh, I forgot. I’ll change the number to 11 and click Update Run Param. That was close.

	Even after the measurement has started, it’s okay. But, it can be concerning if something happens while data is being collected.

I will keep that in mind.

	Mr. Nakamura, we’ve obtained quite good data, haven’t we?

Thank you. But the baseline is sloping upward. It was the same with the water-water measurement. Is this not a problem?

	This slope is considered to occur because the volumes of the sample cell and reference cell are not exactly the same. It is an unavoidable system drift. However, in DSC experiments, the data from the control measurement is always subtracted from the sample measurement data, so if they drift in the same way, they will cancel each other out, and it won’t be a problem.

So the quality of the data is determined by the consistency of this baseline slope, right?

	That’s right.

If the slopes do not match, could it be because bubbles have entered the cell?

	That’s correct. If the baseline reproducibility is poor, first suspect bubbles. The cause of these bubbles could also be due to dirty cells.

	Keeping the cells clean is important for both ITC and DSC, isn’t it!

	Yes, keeping the cells clean is very important. Also, be careful if the composition of the buffer differs, as this can also lead to baseline drift.

	One more thing. It’s important that the sample response is lower than the control response. This time it looks like it is too.

Yes, it is, but… why is that?

	When comparing the control, i.e., the buffer liquid, with the sample solution, it’s considered that the sample has a lower molar concentration of water molecules due to the protein content. Since water has a higher heat capacity than protein, the more it’s contained, the higher the heat emission.

I see. Then, what could be considered if the sample is recorded higher?

	It might occur if there’s a slight difference in the buffer composition.

	Then I’ll wash the system and shut it down.

Good job. Next is the analysis.

	Yes, I will follow the manual for now and proceed.

Oh, I don’t have that icon on my desktop…

	Depending on when you purchased it, you may have a system with the MicroCal Analysis Launcher icon on the desktop instead of the MicroCal, LLC DSC icon. For those, after double-clicking this icon, choose VP-DSC from MicroCal Data Analysis Click button to Start. Subsequent operations follow the manual.

Come to think of it, there are 10 scan data for the control. Which one should I choose? Normally, you should use the control data closest to the sample measurement, right?

Mr. Nakamura, what’s wrong?

There are 10 scans for the control, but in such cases, should I use the 10th scan, which is closest to the sample?

That’s correct. The more you repeat the scans, the better the reproducibility. So in this case, the 10th scan is fine. However, if there was spike noise in the data even in the final control measurement, you would need to choose the one before it.

	Yes, understood. Thank you. Then I’ll call up the two files with the sample on the 11th scan and the control on the 10th scan.
	Note: In the analysis software, it is labeled as Kcal/mole, but it should be kcal/mol.

Mr. Nakamura, it seems like the analysis is going well.

That’s true, but there are two ΔH values. They are approximating each other, but which one should I adopt?

Mr. Nakamura, you asked about that before, didn’t you?

???

The difference between calorimetric enthalpy change (ΔHcal) and Van’t Hoff enthalpy change (ΔHvh).

	Oh, I forgot!

The one with v under ΔH is the Van’t Hoff enthalpy change. Since the values are approximating each other this time, it means RNase A is a protein that transitions in two states.

That’s correct. For this type of sample, it can also be analyzed using a 2-State model.

	By the way, professor. There’s a note in the Test Kit saying “data analysis method: Integrate from 0,” what does this mean?

There’s a function in the analysis software that allows you to find the enthalpy change and Tm using the peak information after subtracting the baseline.

	Let me add that “Integrate from 0” is not mentioned in the Japanese simplified manual. When you perform fitting analysis, data editing is done, but up to the point of baseline subtraction before fitting, the operations are the same. After that, the operations are as follows.

Oh!? Tm and NDH values differ from those during fitting!

While the Tm in “Integrate from 0” seeks the peak top, in fitting, it calculates the temperature at which the peak area obtained through fitting is half. Also, the NDH in “Integrate from 0” refers to actual area, while in fitting, it refers to the area obtained through fitting (the data of the red curve), so the values differ slightly. When you look at the fitting results, there is a deviation between the observed value and the fitting curve, right?

Yes, that is true. By the way, regarding this deviation, what is considered the acceptable range?

When I previously asked someone from Malvern, they mentioned that if the standard error is within 2%, there is no issue.

ΔH is 1.026E5 cal/mol (1.02 kcal/mol), so 2% of that is 2052 cal/mol, which means 409 cal/mol is within the acceptable range.

That’s right.

To add, as per Mr. Nakamura’s previous questions, the decision to obtain parameters via fitting or not is left to the person conducting the measurement. In a 2-state transition, the results from the fitting and “Integrate from 0” are in close approximation because the peaks can likely be symmetric in such samples. Consequently, Tm approximates the peak top. However, in non-2-state transitions, the peaks may not be symmetric, meaning the temperature at the half area doesn’t necessarily coincide with the peak top. The key is to always analyze and compare using the same method.

From the “Download” button below, you can download the “VP-DSC Data Analysis Procedure” mentioned in the text.
(It’s the same as the one available for download in Part 1.)

Download