Michael P. Fuller

Infrared Microsampling by Diffuse Reflectance Fourier Transform Spectrometry

It is shown that diffuse reflectance techniques enable increased sensitivity to be obtained for infrared microsampling compared with the use of KBr micropellets. When nonabsorbing matrices, such as KCl, are used, detection limits of less than 10 ng of samples are observed. Samples absorbed on graphitized substrates, which have a fairly strong general absorption but few intense absorption bands, may also be studied but at somewhat reduced sensitivity. Diffuse reflectance infrared Fourier transform spectrometry does not appear to be particularly useful for studying adsorbates on silica gel, which is not only a strong infrared absorber but also has a surface which is so active that small changes in the surface structure can change the spectrum significantly. Extraction of sample spots from thin layer chromatography plates followed by deposition onto KCl yields much better results than in situ measurements.

Diffuse reflectance infrared spectrometry of powdered coals

Diffuse reflectance Fourier Transform infrared spectrometry has been shown to allow spectra of powdered coal samples to be measured at high sensitivity. Quantitative precision is good, so that the rank of individual coals and the composition of coal blends can be determined in <15 min. The degree of oxidation of coals, both for natural oxidation in the seam and for laboratory oxidation, can be determined from diffuse reflectance spectra of coals and from the second derivative of these spectra. Second-derivative spectra allow quantitative estimates of oxidation to be obtained without the application of spectral subtraction routines, which require standard reference samples that are not always available. The potential for using diffuse reflectance infrared spectrometry for monitoring reactions of powdered coals in-situ has been demonstrated.

FT-IR in Combination with the Attenuated Total Reflectance Technique: A Very Sensitive Method for the Structural Analysis of Polypeptides

We have used Fourier transform infrared spectroscopy to analyze protein structure at nanomolar concentrations and compared its sensitivity with other commonly used spectral techniques such as circular dichroism and fluorescence. Less than 10 nM concentration of protein (immunoglobin G) was required in order to obtain IR spectra with good signal-to-noise ratio that could be utilized for curve-fitting analysis to obtain individual band areas assigned to specific secondary structural features. No signals were observable on circular dichroism, and the fluorescence signals were within the noise level for the same concentration of the protein. The results suggest that FT-IR in combination with the ATR technique has high potential for protein structural analysis, and less than 15 picomole protein is sufficient for the structural analysis.

Molecular structure of tetanus neurotoxin as revealed by Fourier transform infrared and circular dichroic spectroscopy

Secondary structure contents of tetanus neurotoxin have been estimated at neutral and acidic pH using circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. An analysis of the far-ultraviolet CD spectra of the neurotoxin dissolved in 50 mM citrate-phosphate buffer (pH 7.0) revealed 20.0 +/- 2.1% alpha-helix, 50.5 +/- 2.1% beta-pleated sheets, no beta-turns, and 29.5% random coils, which is at considerable variance with results from an earlier detailed study of tetanus neurotoxins secondary structures (J.P. Robinson, L.A. Holladay, J.H. Hash and D. Puett, J. Biol. Chem. 257 (1982) 407). However, the alpha-helix content estimated in this study is consistent with the earlier studies of Robinson et al. (J.P. Robinson, L.A. Holladay, J.B. Picklesimer and D. Puett, Mol. Cell. Biochem. 5 (1974) 147; J.P. Robinson, J.B. Picklesimer and D. Puett, J. Biol. Chem. 250 (1975) 7435) and with the study by Lazarovici et al. (P. Lazarovici, P. Yanai and E. Yavin, J. Biol. Chem. 262 (1986) 2645), although other secondary structural features do not agree with those of the previous studies. Secondary structure estimation from Fourier transform infrared spectra in both amide I and amide III frequency regions revealed 22-23% alpha-helix, 49-51% beta-pleated sheets and 27-28% random coils, indicating a good correlation with the secondary structure content estimated from CD analysis. Lowering of the pH of the neurotoxin to 5.5 or 4.0 did not result in any noticeable change in the overall secondary structures. However, there were significant pH-induced variations observed in the individual curve-fitted FT-IR bands in the amide III frequency region. For example, the 1302 cm-1 band (relative area, 4.2%) observed at pH 7.0 was shifted to 1297 cm-1 (relative area, 2.2%) at pH 5.5, and the relative area of the band at 1316-1317 cm-1 (alpha-helix) increased by approx. 40%. This study suggests that contrary to earlier reports, tetanus neurotoxin is a beta-pleated sheet dominated structure, and although lower pH does not change the overall contents of the secondary structures, significant conformational alterations are observed.

Use of Fourier Transform Infrared/Attenuated Total Reflectance Spectroscopy for the Study of Surface Adsorption of Proteins

The adsorption behavior of lysozyme and immunoglobulin G (IgG) onto a ZnSe crystal surface has been detected by the FT-IR/ATR technique. With this technique we are able to detect the protein adsorption process at a very low concentration (0.0005 mg/mL). The equation of Sperline et al. [Langmuir 3, 198 (1987)] was successfully applied to calculate the adsorption density of protein from an aqueous solution on a real-time basis. The monolayer formation of lysozyme was observed at a concentration range of 10−7 M to 10−5 M. A monolayer-to-multilayer adsorption transition of lysozyme was clearly observed from the adsorption isotherm plot at 35 μM. A differential adsorption density was observed for IgG that could be explained on the basis of its differential size and surface charge.

Fourier transform infrared analysis of amide III bands of proteins for the secondary structure estimation

Protein secondary structure has been analyzed using a Fourier transform infrared spectroscopic method in the amide III region. Although extensive work has been done on protein secondary structure using the amide I region (1700 - 1600 cm-1), the amide III region has not been utilized in the past for its potential in protein structural analysis. One of the major reasons for non-use of the amide III vibrations is perhaps the very weak signal in the amide III frequency region (1200 - 1350 cm-1). However, benefits of using the amide III region are substantial. For example, water vibrations do not interfere with the protein spectrum unlike in the amide I region. In the amide III region, the protein spectrum is better resolved into individual bands than in the amide I region. This feature allows for a greater ease in peak definement of the protein spectra. In the amide III region, the bands for the individual secondary structures ((alpha) -helix, (beta) -sheet and random coils) do not overlap as much as they do in the amide I region. This lack of overlapping allows for easier and a more reliable means of peak assignment, and secondary structure band positions are easier to determine. Amide III region of protein IR spectra appears to be a valuable tool in estimating the amount of secondary structure present in proteins.

Fourier Transform Infrared Spectroscopic Analysis of Proteins in Terms of Detectability, Conformation and Surface Adsorption Density

Publisher Summary Spectroscopic methods commonly used to analyze the polypeptide folding of proteins include fluorescence, circular dichroism, Fourier transform infrared (FT-IR), Raman, and nuclear magnetic resonance spectroscopies. This chapter discusses the use of attenuated total reflectance (ATR) technique for sample handling to test the limits of the sensitivity of FT-IR spectroscopy for protein structural analysis. The use of FT-IR-ATR for the conformational analysis of low concentration proteins can be extremely important to biochemists and molecular biologists who have a minute amount of proteins purified or who have produced small amounts of proteins by genetic engineering. For proteins or protein fragments that are not readily soluble in aqueous solutions, the FT-IR1-ATR approach may provide a useful means to analyze their structure. The technique should also provide a way to analyze the structure of proteins at higher concentrations, which is not possible with circular dichroism or fluorescence. A comparison of protein structure at high concentration such as in precipitate on crystals, and high concentration aqueous solution versus low concentration aqueous solution may provide information about the relationship between structure and biological activity. FT-IR-ATR spectroscopy of polypeptides has a high potential for their conformational analysis at concentration levels that cannot be accurately analyzed by commonly used techniques such as circular dichroism or intrinsic fluorescence.

Cadmium(II)-Exchanged Zeolite as a Solid Sorbent for the Preconcentration and Determination of Atmospheric Hydrogen Sulfide II: Spectroscopic Techniques

Instrumental techniques are compared for usage in the determination of atmospheric H2S which has been preconcentrated on Cd(II)-exchanged zeolite. The study covers the range of 0.01 to 100 μg of immobilized sulfide. For high concentrations (2–100 μg), combustion analysis followed by nondispersive IR is the simplest and most precise technique, and results with the use of this method are accurate to ±3% relative. In the range of 0.25–40 μg, the sulfide, after conversion to methylene blue, may be determined by conventional spectrophotometry, with a relative precision of ±13%. For the lowest concentration range investigated (0.01–2.0 μg), photoacoustic spectroscopy gave the only quantitative results after the H2S was converted to methylene blue.

Sampling And Resolution Enhancement Techniques For The Infrared Analysis Of Adsorbed Proteins.

In this report, we have analyzed the secondary structures of the dichain form of tetanus neurotoxin using. FT-IR and circular dichroic spectroscopies for a-helix, β-sheets, β-turns and random coils. These results indicate that the secondary structures are significantly different from those reported in earlier studies in that it shows much higher content of ordered structures (~50%) which could be significant for the function of the neurotoxin.