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Characterization Quality Control

Easily Determine Protein Secondary Structures IRTracer-100

Analysis of Protein Secondary Structures benefits

—Analysis on Changes of Secondary Structures in Egg White Proteins Caused by Thermal Denaturation— Cell Line Optimization
click here • By using a heatable three-reflection ATR accessory, infrared spectra can be obtained
from proteins in a heated environment.
Operating Principle and Features 40℃
0.06 50℃ • Slight variations in infrared spectral shapes can be shown clearly by calculating the
60℃
70℃
Multiple absorption peaks from C=O stretching vibration of peptide Abs 80℃
90℃
100℃ second derivative of infrared spectra obtained.
bonds overlap to appear as a broad peak near 1650 cm (amide I 0.04
-1
band). Analyzing the peaks can provide information about the protein • The secondary structures of proteins can be analyzed by separating amide I band Culture
0.02
secondary structures. Each absorption band in the overlapping group peaks in second-derivative spectra.
of absorption bands can be determined by a curve-fitting process that
0.00
optimizes peak information (position, intensity, and FWHM) for the 1700 1675 1650 1625 1600
cm -1
curve being fit to each absorption band, so that the difference between Fig. 1 Infrared Spectra of Amide I Band in Egg White
the calculated and measured spectra is minimized. The calculated
spectra are commonly based on the Lorenz or Gaussian curve fitting. Analysis Using Second-Derivative Spectra
The following describes the process for observing the secondary Evaluating second-derivative spectra can be helpful when investigating
structural changes that occur due to thermal denaturation of proteins variations in the secondary structure of proteins (α-helix, β-sheet, β
based on the second-derivative spectrum and peak separation. -turn, and random coil structures). The second-derivative spectrum (Fig.
2) determined from Fig. 1 confirmed that thermal denaturation was Purification
Measurement Method causing an increase in β-sheet structures near 1693 cm and 1622
-1
cm , and β-sheet structures near 1637 cm and α-helix structures
-1
-1
Egg white was used for the sample because it consists primarily near 1655 cm to untangle. The peak shift due to thermal denaturation
-1
of proteins. 60 μL samples were measured using a MicromATR suggests the status of hydrogen bonds may have changed.
measurement accessory with a heatable three-reflection ATR prism
(diamond/ZnSe) installed. Since egg white hardens when heated, the
three-reflection ATR prism was used because it can also be used to
measure solid samples. Due to overlapping between amide I and water
vapor peaks, the optical system was purged with dry air. Given the Decreases
Decreases
measurement conditions in Table 1, a temperature controller was used
to increase the prism temperature from 40 to 100 °C in 10 °C steps, Increases Characterization
with each temperature setting held for two minutes after placing drops Random Increases
of egg white to ensure adequate heat transfer before measuring. To β -Sheet β -Turn α-Helix Coil β -Sheet
eliminate the effects of moisture in the egg white, analysis was based on
difference spectra calculated by subtracting the spectrum for water at Fig. 2 Second-Derivative Spectra of Spectra in Fig. 1
each temperature. Amide I Band Peak Separation Specifications
Based on the peak wavenumber and area value for each amide I
Table 1 Measurement Conditions Instrument IRTracer-100
band peak separated (measurement parameters in Table 2), secondary
Resolution 4 cm -1
structures were attributed to each peak and the ratio of secondary Interferometer Michelson interferometer (30° incident angle)
Accumulation 100
Apodization function Sqr-Triangle structures was determined (Table 3). The resulting tendency for β Equipped with Advanced Dynamic Alignment system Quality Control
Zero filling 4 times -sheet structures to increase and α -helix structures to decrease Sealed interferometer with Automatic Dehumidifier
Detector DLATGS matched the tendencies in the second-derivative spectrum. Optical system Single-beam optics
Beam splitter Germanium-coated KBr for Middle IR (Standard)
Table 2 Conditions for Curve Fitting
Results and Discussion Germanium-coated CsI for Middle/Far IR (Optional)
Peak curve type
Gaussian function
Silicon-coated CaF2 for Near IR (Optional)
Baseline Offset 1 Pt
The difference spectra between egg white and water at each
Range 1710 to 1580 cm -1 Light source High-energy ceramic for Middle/Far IR (Standard) with 3 years guaranteed
temperature showed an increase in prominent peaks near 1625 Max. error 0.01% Tungsten lamp for Near IR (Optional)
-1
-1
cm and 1675 cm at 60 °C or higher temperatures (Fig. 1 is an
-1
enlargement of the 1700 to 1600 cm area). That confirmed its Detector DLATGS detector with temperature control for Middle/Far IR (Standard)
Conditions for Curve Fitting MCT (Hg–Cd–Te) with liquid nitrogen cooling for Middle/Near IR (Optional) Pharmacokinetics
correlation with thermal denaturation.
α-helix β-sheet β-turn Random coil InGaAs for Near IR (Optional)
40 °C 30.3 % 37.9 % 16.4 % 15.4 % -1
Conclusion 100 °C 15.1 % 47.6 % 29.7 % 7.7 % Wavenumber range 7,800 to 350 cm (Standard)
-1
12,500 to 240 cm (Optional)
-1
An FTIR spectrophotometer makes it easy to predict the changes in Resolution 0.25, 0.5, 1, 2, 4, 8, 16 cm (Middle/Far IR)
-1
secondary structures due to thermal denaturation of proteins. It can Application Examples (Shimadzu Application News No.) 2, 4, 8, 16 cm (Near IR)
contribute to protein modification technology, such as improving the Dimensions W 600 mm × D 665 mm × H 295 mm
thermal properties of proteins with a known structure by heating them • Predicting secondary structures in proteins Others
to add structural mutations to structures that are prone to unfolding. • Predicting the locations of mutations in proteins Weight 47 kg
• Evaluation of amyloid-β aggregation (A619)
Measurements Spectrum measurement, continuous measurement, atmospheric correction measurement, continuous
measurement using ASC, simple measurement mode
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