Protein secondary structure analysis in low concentration aqueous solutions

Download PDF August 11, 2022


Infrared spectroscopy is an effective technique for component analysis and interaction analysis of biological samples such as proteins, sugars and lipids. FTIR spectroscopy can be used to measure solid and liquid samples when applied to secondary structure analysis of proteins.

JASCO has previously reported the study of aqueous protein solutions*1.  In this application note, we report results from the ATR PRO PENTA an optimal system for measuring low concentration aqueous solutions. The ATR PRO PENTA has been designed with an optimum number of reflections so that water absorption is not saturated. Furthermore, the use of  an FTIR spectrometer with highly sensitive MCT detector allows measurement of samples with very low sample concentrations, which were previously difficult to measure. Here we report the results of measurement of three examples of low concentration protein aqueous solution.

FT/IR-6800 and ATR PRO PENTA (Overview and Prism image)
Figure 1. FT/IR-6800 and ATR PRO PENTA (Overview and Prism image)

– ATR easy sampling accessory
– Unique pentagonal prism shape
– 14 reflections in a Ge prism (Figure 1)
– High sensitivity measurement in combination with an MCT detector
– Sample volume of only a few microliters


*1 FT/IR Application Note: 210-TR-0127


Analysis of low concentration protein aqueous solution

The following three protein aqueous solutions (0.01% w / v) were prepared and IR spectra measured using an ATR PRO PENTA. Secondary structure estimation (SSE) was performed by taking the difference spectrum with water.

1: Lysozyme (Chicken Egg White)
2: Concanavalin A
3: Trypsin inhibitor Kunitz

Measurement Conditions
Main UnitFT/IR-6800Accumulation90 times
AccessoryATR PRO PENTAResolution4cm-1
DetectorMCT (PV type)

Figure 2 shows the IR spectrum of 0.01% Lysozyme aqueous solution and the difference spectrum with water. In the difference spectrum, protein amide I and amide II bands were clearly observed.

Figure 2. 2 IR Spectrum of 0.01% aqueous Lysozyme (left), difference spectrum (before ATR correction) (right)

The SSE result of the amide I band of the ATR-corrected difference spectrum and the reference value*2 by X-ray diffraction are shown. Each sample was measured in triplicate and all were in good correlation with the reference value. From this result, it was shown that ATR PRO PENTA is effective*3 for analysis of low concentration protein aqueous solutions.

*2 Sarver, R. W., Krueger, W. C., 1991. Anal. Biochem., 194, 89-100.
*3 Depending on the type of protein, it may adsorb on the prism and affect the results



Secondary structure estimation, low concentration, ATR, multiple bounces, MCT detector, proteins


Figure 3. Result of SSE analysis of three protein aqueous solutions (0.01% w/v)
ModelDescriptionPart Number
Main UnitFT/IR-6800FT/IR Spectrometer7085-J006A*1
DetectorMCT-6000PVMCT(PV) detector6584-J345A*2
AttachmentATR PRO PENTAMulti reflection ATR6584-J343A
SoftwareIR SSE-4000Secondary structure estimation program4880-7234A

*1 FT/IR-4600/4700 and FT/IR-6600/6700 are also available.
*2 When using FT/IR-4600/4700, MCT-4000PV should be used.

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About the Author

Dr. Carlos Morillo received his Post Doc at Advanced Industrial Science & Technology in Fukuoka and was a Research Scientist at Kyushu University in Japan where he lived for several years. Carlos received his Doctor of Engineering from Kyushu University and his Masters and BS from Simon Bolivar University in Caracas Venezuela. He is an Applications Scientist at JASCO.