Amide III region

Further evidence for these a-helix ROA band assignments in the extended amide III region comes from the ROA spectrum of poly-L-alanine dissolved in a mixture of chloroform (70%) and dichloracetic acid (30%), known to promote a-helix formation (Fasman, 1987), which shows strong positive ROA bands at 1305 and 1341 cm-1 (unpublished results), and of the cv-helix forming alanine-rich peptide AK21 (sequence Ac-AAKAAAAKAAAAKAAAAKAGY-NHg) in aqueous solution which shows strong positive ROA bands at 1309 and 1344 cm-1 (Blanch et al., 2000). 

In their initial studies, Tfayli et al. [33] acquired spectra from an Episkin model. This model is comprised of human adult keratinocytes which produce stratified epidermis following a 13 h culture period. Raman spectra from this model were compared with normal human skin. Significant differences were noted, particularly in spectral features arising from the 850/830 tyr Fermi doublet (which is sensitive to the H-bonding state of the -OH group [34]) and in the protein amide III region. Usable spectra were acquired to a depth of 15-20 pm. 

The first approach is illustrated in Figures 4, 5, and 6. Figure 4 shows spectra of y-globulin (top), albumin (bottom), and a 1 1 mixture of albumin and y-globulin (middle). The presence of both components could possibly be deduced from the spectra of the mixture by both the bands in the 1400-1450-cm-1 region and by the bands in the Amide III region. The ratio... 

Previous infrared studies indicate that proteins with a large amount of 3-sheet structure absorb near 1240 cm and those with a-helix stucture absorb near 1280 cm l (22,32). Absorbances for denatured albumin, reportedly containing random and 3-sheet conformations, are found at 1240 and 1260 cvT (22). These assignments correlate with the more studied Raman spectroscopy of the amide III region which has vibrations at 1230-1250 cm l for 3-sheet structure, at 1260-1290 cm for a-helix structure, and at 1240-1265 cm l for unstructured polypeptide (34,35). 

Figure 8. Absorbance spectra of the amide III region of solution FN (A), and FN adsorbed on PEO-PEUU (b), PDMS-PEUU (C) and PTMO-PEUU (D), Each spectrum is not from a single experiment, but is the co-addition of 4 spectra on a given polymer at 2 hours of adsorption.

The FTIR/ATR studies of FN adsorption indicate that the polymer surface plays an important role in determining both the amount and the conformation of adsorbed FN, Comparison of the PEO-PEUU surface with the PDMS-PEUU and PTMO-PEUU surfaces show that on the more hydrophilic PEO-PEUU polymer, less protein adsorbs, and the interactions between the protein and polymer take place more slowly and are less intense as shown by the late appearance of the COOH vibration and the smaller 1669 cm l amide I peak observed at early adsorption times. The protein which adsorbs first appears to interact most strongly with the surface. On all polymers, changes in the amide I and the amide III region of adsorbed FN suggests that the amount of B sheet structure in FN increases upon adsorption. 

From the spectra shown in the figures and following the discussion in the text the secondary structure assignments for the five groups of proteins are summarized in Table 2. From this table is can be observed that for the Amide I vibration the major structural components dictates the Amide I frequency, but it also indicates that one structure influences the frequencies of the other. For example, the a-Amide I frequency falls at 1656 cm , but as more and more B-structure is present the frequency shifts from 1656 cm (a ) to 1652 cm l ( a+ low B ) to 1644 cm l (a + B ). In the Amide III region the a-helix frequency shifts from 1298 cm to 1315 cm whenever some B Sheet structure is present. Similarly, the B-sheet Amide III shifts from 1235 cm" to 1241 cm" when enough a-helix structure is present. 

Structural changes in proteins, e.g., resulting from microenvironmental factors such as pH or from protein-protein interactions, can be observed in the IR (especially in the conformation-sensitive Amide III region), also without requiring added probes of the protein structure. 

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