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.

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.

instrument manufacturer to provide a more turn key Vl'-Raman system has become apparent. It is nowappropriate to examine several of the spectroscopic operations which are routine for the FT4R experiment asthey relate to the FT-Raman experiment. Some of these operations include the ability to create and searchspectral databases for qualitative analysis and to use commercially available chemometric methods forquantitative analysis.Quantitative analysis by Raman spectroscopy using external standards has proven difficult owing to theeffects of instrumental parameters which can alter the reproducibility of band intensities from scan to scan.However, near-infrared FT-Raman spectroscopy has several advantages over the traditional dispersivetechnique which may make quantitative FF-Raman feasible. 1,2 One principal advantage of the VF-Ramantechnique is the fact that the entire spectrum is sampled with each scan, and each scan can take less than onesecond to acquire. Thus, the FT-technique may be less sensitive to instrument drift as these effects areaveraged over the entire spectrum. In addition, the entrance aperture of the interferometer system is large,making the sampling geometry less sensitive to absolute repositioning of the sample cell.1'2 Finally, theresolution and throughput are constant across the entire spectral range. These features mean that spectrawith a high degree of reproducibility can be acquired using very simple experimental geometries.The test of subtraction in the ET-Raman serves to verify both the reproducibility of the sampling as well asthe linearity of the detection system. In this test, a spectrum obtained by subtraction of the two alternateisomers of xylene from the isomer of interest can be directly compared with that obtained from the neatsample of the isomer. It is of particular interest to examine the areas of significant band overlap, where thequestion of detector linearity is key. Figure 1 shows the results obtained on the first set of mixtures of the

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.

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.

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% α-helix, 50.5±2.1% β-pleated sheets, no β-turns, and 29.5% random coils, which is at considerable variance with results from an earlier detailed study of tetanus neurotoxin's secondary structures (J.P. Robinson, L.A. Holladay, J.H. Hash and D. Puett, J. Biol. Chem. 257 (1982) 407). However, the α-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 22–223% α-helix, 49–51% β-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 (α-helix) increased by approx. 40%. This study suggests that contrary to earlier reports, tetanus neurotoxin is a β-pleated sheet dominated structure, and although lower pH does not change the overall contents of the secondary structures, significant conformational alterations are observed.

FT-IR spectrometers designed for laboratory operation are generally not suited for plant production areas where ambient temperatures often exceed 120° F and noise levels are extremely high. The Nicolet Pro-IR 5 process FT-IR spectrometer can operate in hostile environments over the spectral range from 10 000 to 250 wavenumbers. We have evaluated the performance of this system using a set of commercial detergent samples and a wide range of gas mixtures. The detergent samples were simultaneously analyzed for twelve components using a sophisticated partial least-squares statistical analysis software package. The concentrations ranged from 0.1 to over 50 wt.-%, with water being the major component. A comparison will be made between the NIR and MIR results using flow-through ATR (attenuated total reflectance) and transmission cells.

An implementation of the PLS (partial least-squares) statistical approach to quantitative analysis was applied to a set of mid-infrared spectra obtained from a series of commercial detergent samples. The components analyzed included the base detergent, sodium benzoate, 2-propanol, 1,2-propanediol, polypropylene glycol, and glycerol. The samples were analyzed with the use of a horizontal attenuated total reflectance (ATR) accessory equipped with a zinc selenide crystal. The PLS model created in the calibration was found to provide excellent results for a set of validation samples.

Various approaches to infrared multicomponent quantitative analysis including K-matrix, multivariate least-squares, principal component regression (PCR), and partial least-squares (PLS) are compared. The advantages and disadvantages of each are discussed. A particular implementation of the PLS method is detailed, with emphasis on the methods provided for calibration optimization and evaluation.

Quantitative photoacoustic and transmission infrared spectroscopy have been compared for a highly overlapped three component mixture with concentrations varying from 0 to 60%. A partial leastsquares model has been employed for the quantitative analysis. High correlation coefficients were obtained for both the transmission and photoacoustic model.