The Path to Becoming a Calorimeter Master Vol.1 (Continued) What is a Microcalorimeter?

by Malvern Panalytical, Monday, 15th June 2015

Nakamura: “In the seminar materials, the overviews and examples of ITC and DSC are briefly summarized, and I vaguely understand what kind of data can be obtained and what information can be derived from there. However, it seems that there is no explanation on how to actually measure… First, let’s investigate ITC. iTC200…”

Malvern’s MicroCal iTC200… so this is it…

MicroCal iTC200 Website

*The iTC200 was discontinued as of March 31, 2017.
Please refer to the currently available PEAQ-ITC.

Oh, there is also a movie. When a reaction between samples in the sample cell generates heat, a temperature difference occurs with the reference cell, which is controlled to eliminate that difference… I see. So when exothermic signals appear downwards to lower the sample cell temperature, that’s what it means. I get it. But this seems to be greatly affected by the outside air. I wonder if air conditioning is okay?

There seems to be an explanation of the principle as well.

Data often seen in papers. But why can dissociation constants be determined from this data?
Is KD determined by the tangent?

ΔH, enthalpy, is the change on the vertical axis, right? Hm? The unit of the vertical axis has changed. It shows the heat change per 1 mol of the titrated sample.

I wonder if it’s written in more detail on the technical page?
Let’s have a look.

Isothermal Titration Calorimetry Technical Page

“Ultimately, each peak is integrated and plotted against the molar ratio of ligand to protein. The resulting isothermal titration curve is fitted with a binding model where the affinity (KD) can be obtained.” So they are not determining the dissociation constant from the tangent but fitting it with the theoretical formula.

“The molar ratio at the midpoint of the isothermal titration curve determines the binding ratio.” But does this mean how many binding sites there are in the sample on the cell side? Why is the binding ratio determined by the molar ratio at the midpoint?

Enthalpy change was the change on the vertical axis, but here it says “Binding mechanism.” Why is enthalpy a mechanism? And entropy is not mentioned!

Hm, I don’t understand…

But it seems that sample preparation is easier because there’s no need for labeling or immobilization. “When a ligand binds to a protein, a change of one-millionth of a degree occurs,” but it’s a device that detects such a small temperature change. Well, it would be a problem if the body temperature changed so much upon binding in the body.

I wonder in what fields ITC is useful…?

“ITC is widely used in drug discovery and development for the following purposes.” It says…

“Quantification of binding affinity” – This means obtaining parameters via ELISA, RI, SPR, etc., right? No immobilization or labeling, and no washing, so reactions in a more natural state can be obtained, which is an advantage, I suppose?

“Selection and optimization of drug candidates” – Does this mean screening based on binding strength?

“Measurement of thermodynamics and active concentration” – Thermodynamic parameters are useful information in structural analysis, but what is this active concentration measurement? Could it be the “protein quality control” mentioned in the Interphex materials?

“Characterization of mechanism of action” – I’m not sure what this means. Could it be the mechanism of binding?

“Identification of specific targets for small-molecule drug discovery” – Is this screening? But the throughput doesn’t seem very high…

“Understanding binding specificity and binding ratio” – How do you distinguish non-specific binding?

“Validation of IC50 and EC50 in hit-to-lead” – Does this refer to the optimization post-screening?

“Measurement of enzyme reaction rates” – Enzyme!? Can it measure such a thing?

Or is it that I can’t understand exactly how to measure it? Can’t I download manuals or something?

For now, let’s contact the manufacturer.”

So, it seems Nakamura-san has quite gotten into microcalorimetry.

It seems like he is trying hard on his own, but there keep being things that cannot be resolved with just the website or manual.

From now on, almost every month, we will introduce the story of Nakamura-san until he masters calorimetry.

We also plan to provide helpful Q&As and troubleshooting for beginner and intermediate calorimetry users.

Stay tuned!!

We are also releasing related technical materials. Please download and make use of them.

By filling in the required fields in the form, you can download the following set of materials.

ITC Application Notes

DSC Application Notes

Back Issues of The Path to Becoming a Calorimeter Master

Introduction of Dr. Harumi Fukada
Visiting Researcher at Osaka Prefecture University
[Brief Career History]
Graduated from the Department of Agrochemistry, School of Agriculture, Osaka Prefecture University in 1974
Withdrew after earning credits at the Graduate School of Agriculture, Osaka Prefecture University in 1979
That same year, became an Assistant at the School of Agriculture, Osaka Prefecture University
Associate Professor at the School of Agriculture, Osaka Prefecture University in 1997
Associate Professor at the Graduate School of Life and Environmental Sciences, Osaka Prefecture University in 2008
Retired in 2015

Blog Epilogue

Dr. Fukada has been involved in building calorimetry systems under Dr. Katsutada Takahashi at Osaka Prefecture University’s School of Agriculture, back when biochemical calorimetry hadn’t spread much in Japan. In the early days, she used Dewar-type calorimeters and flow-type calorimeters, consuming tens of mg of protein at a time. Her laboratory was also the first in Japan to measure proteins from a biochemical standpoint. She is among the few who have used most of the older models of DSC and ITC, including the first Privalov-developed DSC for protein solutions in the world. Dr. Takahashi continues his research on measuring the heat generated by microbes as part of JST’s commissioned work, and develops new “bioactivity measuring devices,” even after turning sixty.Download

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