Microstructural study of thermal denaturation and gelation of proteins using an Agilent 660 FTIR

Significance of the Topic

The thermal denaturation and gelation of proteins underpin many processes in food science, biopharmaceutical formulation, and industrial biotechnology. Monitoring microstructural changes in real time helps optimize product texture, stability, and functionality, making analytical methods that reveal molecular transitions essential for research and quality control.

Objectives and Study Overview

This study employs mid-infrared Fourier transform spectroscopy to track structural alterations in β-lactoglobulin A across a broad temperature range. The primary goal is to elucidate how secondary structure elements respond to heating and to correlate spectral signatures with protein denaturation and aggregation phenomena.

Methodology and Instrumentation

A 5% (w/v) solution of β-lactoglobulin B was prepared in deuterated phosphate buffer (pH 7) and heated from 40 to 92 °C in a temperature-controlled transmission cell. An Agilent Cary 660 FTIR spectrometer, operating in rapid-scan mode (5 kHz, 128 scans per spectrum, 4000–800 cm⁻¹ range, 4 cm⁻¹ resolution), collected time-resolved spectra. Data processing and kinetic analysis were performed using Agilent Resolution Pro software.

Main Results and Discussion

Deconvolution of the amide I band at 40 °C revealed seven components assignable to β-type structures (1691 cm⁻¹), β-sheet (1677, 1634 cm⁻¹), turns (1664 cm⁻¹), α-helix (1648 cm⁻¹), β-strand (1622 cm⁻¹), and side-chain vibrations (1614 cm⁻¹). Upon heating above 76 °C, bands at 1677, 1648, 1634, and 1614 cm⁻¹ disappeared, indicating loss of native secondary motifs, while new peaks at 1682 and 1617 cm⁻¹ emerged, characteristic of intermolecular antiparallel β-sheet aggregates. These spectral shifts map the unfolding to aggregation pathway and identify hydrogen-bonding changes during gelation.

Benefits and Practical Applications

• Real-time monitoring of protein folding, unfolding, and aggregation under controlled heating.
• Quantitative correlation between spectral features and physicochemical transitions.
• Enhanced understanding of whey protein functionality to improve food texture and stability.
• Guidance for process optimization in industrial protein formulation.

Future Trends and Potential Applications

Advancements may include coupling FTIR with multivariate analysis and molecular modeling for predictive insights, integration into high-throughput or inline process analytical technology, extension to complex protein mixtures or membranes, and exploration of dynamic structural phenomena under varying environmental conditions.

Conclusion

The Agilent Cary 660 FTIR spectrometer effectively captures rapid structural transformations in β-lactoglobulin during thermal treatment. The method provides detailed molecular insights into denaturation and aggregation, offering a valuable tool for research and industrial quality control in protein-based systems.

Reference

Ismail A, Kirkwood J. Microstructural study of thermal denaturation and gelation of proteins using an Agilent 660 FTIR. Agilent Technologies Application Note. 2012;5991-0019EN.