Protein combination bands

All the higher (1st through 6th) overtones of the 0-H, N-H, C-H, and S-H bands from the IR are seen in the(much smaller) NIR region. This, in addition to combination bands (e.g., C=0 stretch + N-H bend in protein), gives rise to a crowded spectrum with severely overlapping bands. 

The absorption intensity of the 0-1 vibronic sideband is entirely borrowed by HT coupling. The observation that this 0-1 transition is of comparable intensity to the 0-0 transition suggests the feasibility of resonance Raman processes in which both the absorption and emission moments are borrowed. This second-order HT coupling displays itself in the appearance of first overtones and combination bands of non-totally symmetric modes. These are observed extensively in the haem proteins 35). 

The amide I band has been examined by Elliott et al. (1950) in native and denatured insulin, by Elliott et al. (1957) in lysozyme, and by Ambrose et al. (1951) in water-soluble silk. The band at 3200 cm" has also been investigated. Beer et al. (1959) have given a comprehensive list of proteins studied up to 1959, along with characteristic absorption bands. Bamford et al. (1956) have reviewed work done up to 1956 in the region between 5000 and 4500 cm (combination band of the N—H stretching frequency and that of the amide I or amide II band). The infrared dichroic properties of crystals of hemoglobin and ribonuclease have been observed in this region (Elliott and Ambrose, 1951 Elliott, 1952). 

In 1967 to 1969 a series of reports appeared in which it was shown that the mRNA in polysomes is also combined with proteins (Weisberger and Armentrout, 1966 Huez et al. 1967 Henshaw, 1968 Perry and Kelley, 1968 Temmerman and Lebleu, 1969). The essential experiment consisted of the following steps the polysomes were isolated and treated with EDTA to dissociate them into the ribosomal subunits and mRNA. Then the dissociated material was fixed by formaldehyde and ultracentrifiged in a CsCl density gradient. mRNA was banded separately from ribosomal subunits and occupied a rather wide zone with buoyant density of 1.40 to 1.50 g/cm (with the majority of the material at 1.45 g/cm ) from which it is evident that this mRNA was not in a free state but combined with some protein. 

Commercial grains are commonly analysed by using NIR spectroscopy [12, 15]. The major constitnents of grains are water, protein, oil, fibre, minerals and carbohydrates and it is commercially important to quantitate the composition. The NIR spectra of snch materials are, thus, dominated by the overtones and combination bands of C-H, N-H, O-H and C=0 bonds. Figure 8.16 illustrates the NIR spectra (in reflection mode) of a number of common grains, i.e. sesame seeds, sunflower seeds, wheat, barley, grass and oats. 

Both the oxidation-reduction potential and the fluorescence of flavin nucleotides are modified profoundly by attachment of the nucleotide to various proteins. Flavin enzymes have been reported to have oxidation-reduction potentials at pH 7 ranging from —0.4 to 0.187. The combination to proteins also results in shifts of the absorption maxima. The 450 m u band is found at 451 mju in Straub s diaphorase and at 455 m/t in Haas yellow enzyme, while the 375 m/t band appears at 359 m/t and 377 m/t in these preparations. Most flavin enzymes do not fluoresce, and it is assumed that the quenching of fluorescence implies binding of the flavin to the enzyme through N-3. Straub s diaphorase, unlike most other flavoproteins, does fluoresce. This may be evidence that this diaphorase is a partially degraded cytochrome reductase. 

Some useful data on proteins can also be obtained from the overtone region, especially as it them becomes possible to work in the presence of liquid water. These absorptions also show dichroism, but it must not necessarily be assumed that this will be always in the same direction as the parent fundamental, and many of the bands in this region are combination bands arising from more than one fundamental. Thus a band near 4600 cm" in many proteins was originally identified with the carbonyl absorption [53] as it shifts with alterations of the helical form and is present in polymethylmethacrylate but absent in polyethylene. However, this has recently been shown by Hecht and Wood [141] to be a combination band of... 

The main combination bands of secondary amides are summarized in Table 8.5. These bands are important in the analysis of proteins. As explained by Murray in his chapter on spectral comparisons,24 two of the combination bands of proteins lie on either side of the OH combination band of carbohydrates. Therefore, the shape of the spectral region near 4760 cm- (2100 nm) is... 

Elliott and Ambrose identified absorption bands for polypeptides and proteins at approximately 3505 cm- (2853 nm) as N-H-associated stretching bands and at 4825 cm- (2073 nm) in the overtone region. The main result of their work was to demonstrate that a band at 4840 cm" (2066 nm) is useful for distinguishing the presence of extended vs. folded configurations for polypeptides and proteins even in the presence of liquid-phase water. The band near 4824 cm- (2073 nm) was classified earlier by Glatt and Ellis on work in nylon as a combination band of the N-H deformation and stretching modes. 

N-H [vN-H and amide II deformation (N-H in-plane bending) combination] for secondary amides in proteins CONHj combination of amide A and amide II N-H stretching and C=0 stretching (amide I) combination N-H (vN-H and 5N-H combination) for gamma-valerolactam N-H combination band found in the spectrum of native RNase A (C=0 amide I band)... 

N-H from CONH2as thermal unfolding of RNase A protein in aqueous solution (assigned to an N-H combination band)... 

N-H combination band from secondary amides in proteins N-H [vN-H and amide 11 deformation (N-H in-plane bending) combination] for secondary amides in proteins N-H from protein... 

O-H related combination from water change in phase and N-H/C-N combination band from urea (NH2-C=0-NH2) from ovalbumin O-H related combination from water change in phase with the increase in protein concentration at Ovalbumin protein... 

C-H methylene C-H, associated with ovalbumin protein side chains seen at pH 5.0 C-H (2VCH2 symmetric stretching and SCHj) combination band from ovalbumin protein side chains seen at pH 5.0 C-H from ovalbumin protein side chains... 

C-H (2vCH2 symmetric stretching and 8CH2) combination band from ovalbumin protein side chains seen at pH 5.0... 

N-H combination band from secondary amides in proteins 2060 4850... 

N-H (vN-H and 8N-H combination) for NH, (ammonia) in water N-H (3v) for NH5 (ammonia) in water N-H from CONHj as thermal unfolding of RNase A protein in aqueous solution (assigned to an N-H combination band)... 

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