Quantifying Differences in the Secondary and Tertiary Structure of Enzymes

Circular dichroism (CD) spectroscopy is an analytical technique that is used to measure the CD of molecules, which describes the difference in the absorption of left-handed circularly polarized light (L-CPL) and right-handed circularly polarized light (R-CPL) of molecules containing one or more chiral light-absorbing groups, over a range of difference wavelengths. While this technique is used extensively to study a variety of chiral molecules, Chirascan spectrometers have been shown to surpass the conventional use of CD spectrometers. The Chirascan is a highly specialized derivative of traditional CD spectrometers, and Table 1 provides how this instrument provides high-quality data on the higher order structure (HOS) of complex molecules.

Table 1. Data provided by the Chirascan spectrometers.

HOS Information Secondary Structure Tertiary Structure
Wavelength Range Far- UV (<250 nm) Near-UV (>250 nm)
Chromophores Peptide Bonds
S-S Bonds
Aromatic Amino Acids
S-S Bonds
Information Overall conformation
Turns, etc.
Local conformation
Side-chain environment

Effect of Transient Exposure to Nanoparticles on Secondary Structure and Stability of Proteins

Sample Preparation and CD Analysis

Two globular enzymes submitted by a leading European university for analysis were initially incubated with aluminum oxide nanoparticles (NPs) for one hour. The enzyme and NP solution was then centrifuged to remove the NPs and the supernatant was analyzed by a Chirascan V100 spectrophotometer. The following measurements were taken:

  • Far-UV
  • 0.5 mm pathlength cuvette at 20° C
  • Sample concentration 0.3 mg/mL

Additionally, thermal denaturation data was measured using a continuous multiwavelength temperature ramps from 20° C to 90° C were taking at a heating rate of 1° C per minute, to thereby generate 71 spectra in a 71 minute duration.

Data Analysis

A global fit of multiwavelength data was used to analyze the thermal denaturation data.

Results: Exposure to Nanoparticles Modifies Secondary Structure of Enzyme 2

Figures 1-4 show the results of the enzymes analyzed in this study.

Figure 1. Far-UV spectrum for Enzyme 1, which is typical for a predominantly α-helical protein that has some β-sheets.

Figure 2. Far-UV spectrum for Enzyme 2, which is typical for globular protein that is dominated by β-sheets in a distorted conformation.

Figure 3. The effect of aluminum oxide NPs, which show a minor change in far-UV that is indicative of a minimal effect on the secondary structure of Enzyme 1. This spectra was normalized by the absorbance.

Figure 4. The effect of aluminum oxide NPs, which show a signficiant change in far-UV that is indicative of a perturbation effect on the secondary structure of Enzyme 2. This spectra was normalized by the absorbance.

Results: Exposure to Nanoparticles Alters Enzyme 2 Protein Stability

Figures 5 and 6 show how the aluminum oxide NPs altered the protein stability of Enzyme 2. The use of the Chirascan V100 in this experiment provides information for the changes that occur to both protein stability and secondary structure.

Figure 5. The control of Enzyme 2. The signal at 231 nm shows a reduction upon unfolding, whereas there is an increase and shift in the signal at 203 nm.

Figure 6. A modified unfolding of Enzyme 2 shown following NP exposure, in which the enzyme experiences an increase in melting temperature.

Statistically-Validated HOS Comparisons of NIST mAb Variants

Sample Preparation and CD Analysis

Both the reference material utilized in this experiment which was of a lower monomeric purity (RM 8671), and the primary monoclonal antibody standard (PS 8670) were provided by the National Institute for Standards and Technology. Both RM8671 and PS 8670 were dialyzed against PBS, in which the dialysate was used to get a baseline spectrum.

All isothermal CD measurements were carried out by the use of a fully integrate Chirascan Q100 for HOS analysis. The following HOS analysis was performed:

  • Far-UV: six independent replicates and 0.1 mm pathlength flow cell
  • Near-UV: five independent replicates and 10 mm pathlength flow cell

Data Analysis

The weighted spectral difference (WSD) method was used in this experiment, in which the data was computed to generate a quality attribute for statistical analysis. The analysis of this attribute was performed using a quality range approach in which the acceptance criteria of +/-2SD was recommended for intermediate (tier 2) risk ranking. The following equation was used to determine the WSD data.

Results: Minor Differences Detected in Far- and Near- UV

Figures 7 and 8 show both the far- and near-UV spectra of this experiment.

Figure 7. Far-UV spectra of mAbs that is typical for antibodies (Left). Shaded area is the 2xSD envelope (Right).

Figure 8. Near-UV spectra of mAbs that shows the aromatic side chains contributing to their own characteristic wavelength profile (Left). Shaded area is the 2xSD envelope (Right).

Results: Statistical Significance of Minor Differences in Secondary and Tertiary Structure Determined

Figure 9. Tier 2 quality approach that was applied to +/-2SD acceptance criteria. No significant differences in the secondary structure were significant, whereas significant differences in the tertiary structure were shown.


The use of the Chirascan Q100 in this study shows that this tool allows for optimal detection and objective statistical quantification of minor differences in both the secondary and tertiary structure of complex molecules as compared to traditional CD spectrometers.

This information has been sourced, reviewed and adapted from materials provided by Applied Photophysics.

For more information on this source, please visit Applied Photophysics.

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