Using the ConcentratIR2 to Analyze the Bovine Gamma Globulin

Recently, infrared spectroscopy has been recognized as a practical method for protein structure analysis due to its high information content for biological systems. Increased focus has been given to the application of infrared spectroscopy for the analysis of protein unfolding.

The protein unfolding process has been analyzed widely by other techniques such as circular dichroism, fluorescence, or visible absorption spectroscopy. However, infrared spectroscopy is a unique method that enables the analysis of the protein unfolding process through secondary structure modification on a timescale unattainable by other methods, while retaining remarkably high structural resolution.

Attenuated total reflectance (ATR), with its easy sample preparation and high sampling rate, has been established as an advantageous infrared method for testing proteins. The Harrick ConcentratIR2™ is a multiple reflection ATR accessory (Figure 1) designed to examine small quantities of liquid on a timescale small enough for effective analysis of the protein unfolding process.

This article analyzes the application of the ConcentratIR2™ to test small quantities of protein experiencing secondary structural alteration due to thermal perturbation.

Figure 1. The Harrick ConcentratIR2™ multiple reflection ATR accessory

Experiment

The unfolding of BGG protein assay standards (Figure 2) is examined in this research. The solution included a mixture of 750 ug/mL BGG in a 0.9% sodium chloride solution and sodium azide. Measurements were performed using a FTIR spectrometer that is commercially available in the market and fitted with MCT detector and KBr beam-splitter. A multiple reflection silicon ATR element was used to configure the ConcentratIR2™.

Figure 2. The ATR spectra of BGG at 100°C at 0 (lowest peak), 1, 2, 3, 4, 5, 6, 7, 8, 9, 20, and 30 (highest peak) minutes after injection of sample into cell.

Data over a wavelength range of 4000 - 650 cm^-1 was collected. Data collection Parameters were as follows:

Aperture was set to 100%, gain was set to eight, number of scans was 64, and resolution to 4 cm^-1. The ConcentratIR2™ was linked to its heated liquid cell, which was modulated by a Harrick Temperature Controller. The temperature of the heated cell was maintained at 100°C throughout data collection.

With the temperature remaining constant at 100°C, protein samples were injected by syringe through the Luer fittings on the heated cell. An adequate amount of sample was loaded to make sure that the cell interior is fully filled. The spectrum was collected instantly upon injection into the cell and then every minute for nine minutes. Two extra spectra were run 20 and 30 minutes after inspection. The cell fittings were capped during data collection to avoid evaporation.

Results

Vivid spectral changes in relation to one another were exhibited by the collected spectra, demonstrating that protein unfolding was happening due to a significant spike in environmental temperature. Alterations in the characteristic amide I band about 1600 -  1700 cm^-1 were the primary focus.

In this region, the peaks provide maximum data concerning the structure of the protein backbone, which is the main determinant of the protein secondary structure. Inside this region, a concomitant increase in intensity of absorption and wavelength of absorption is noticed over time.

The largest, most striking shift in wavelength - a shift from approximately 1670 cm^-1 to 1620 cm^-1 - is witnessed between the time of injection (t=0) and one minute after injection (t=1). Conversely, the intensity of absorption did not alter greatly during this period. This indicates that the greater part of the secondary structure change could have happened directly after exposing the protein to heat.

As time advances, the spectra appeared to reverse this preliminary trend-increasing in absorption, but staying quite constant in peak position. The spectra acquired every minute from two minutes demonstrate an increase in absorption but a slightly reduced increase with each passing minute.

Comparing these to the spectra collected outside this period (t=20 and t=30) provides a better understanding about protein unfolding in the longer-term. These later spectra are almost identical in intensity and peak position in the amide I region, signifying that protein conformation is not greatly modified beyond a specific time period.

These results create potential for future studies which could involve the use of ConcentratIR2™ to produce a highly comprehensive profile of the rate of secondary structure change linked with thermally induced protein unfolding.

Alterations in base protein conformation caused by heating lead to certain known alterations in secondary structure composition that generate characteristic peaks in the infrared. The amide I band are made up of overlapping component bands that imitate specific secondary structures such as alpha helices, turns, beta sheets, and random structures.

Within the amide I region, globular proteins are likely to generate prominent bands corresponding to particularly beta sheets and alpha helices. These bands constantly fall at wave numbers approximately 1660 cm^-1 and 1620 - 1640 cm^-1 for alpha helices and beta sheets, respectively with minor variation possible based on the specific protein in question.

Analysis of the t=0 spectrum shows an obvious alpha helix peak, ranging from 1670 to 1650 cm^-1; while there is minimal contribution from beta sheets. As illustrated before, the most crucial change in secondary structure appeared to happen in transition from t=0 to t=1.

Based on this transition, there is a noticeable disappearance of the previously mentioned alpha helix peak and a concomitant appearance of a major beta sheet peak about 1620 - 1640 cm^-1, suggesting a transition in the total secondary structure composition to mostly beta sheets and matching with the results of another infrared study that also witnessed an increase in beta-sheets and a decrease in alpha-helices following thermal perturbation.

Conclusion

The article discusses the simplicity and efficiency that the ConcentratIR2™ can visualize thermally induced secondary structure alteration in small quantities of a protein such as BGG. The above results show that when compared to other techniques of protein visualization, IR spectroscopy can achieve a high level of structural detail, especially in recording minute-to-minute alterations in structure linked with protein unfolding.

This information has been sourced, reviewed and adapted from materials provided by Harrick Scientific Products, Inc.

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