Nucleotide absorption maxima

Like the nicotinamide coenzymes (Fig. 13-15), the flavin nucleotides undergo a shift in a major absorption band on reduction. Flavoproteins that are fully reduced (two electrons accepted) generally have an absorption maximum near 360 nm. When partially reduced (one electron), they acquire another absorption maximum at about 450 nm when fully oxidized, the flavin has maxima at 370 and 440 nm. The intermediate radical form, reduced by one electron, has absorption maxima at 380, 480, 580, and 625 nm. These changes can be used to assay reactions involving a flavoprotein. 

The spectra of a free base and the corresponding nucleoside are quite similar, since only the purine or pyrimidine base component is a chromophore, giving rise to absorption in the region between about 250 and 270 nm at neutral pH. Because of protonation of the base nitrogen atoms, spectra of these compounds are also highly pH-dependent Nucleic acids have an absorption maximum close to 260 nm. The variation in absorption coefficient of DNA and RNA per nucleotide residue is small hence the absorption at 260 nm can be used as a measure of total nucleic acid concentration (7). However, the absorption of a native nucleic acid molecule cannot be expressed as a sum of absorbances of all the... 

The molecular weight of 320,000 obtained for the muscle enzyme from sedimentation-diffusion data at 2-6 mg/ml and v = 0.75 (132) is to be compared with 270,000 obtained by Wolfenden et al. from s20,w = 11.1 S and D2 ,w = 3.75 X 10 7 cm2 sec1, and v = 0.731 calculated from the amino acid content (92). The rabbit muscle enzyme has a normal amino acid content, that is, no unusually low or large amount of a particular amino acid was found. Of the 32 cysteine/half-cystine residues per mole based on a molecular weight of 270,000, 6.2 were rapidly titrated with p-mercuribenzoate (92). Typical protein absorption spectra were reported for elasmobranch fish (126), carp (125), rat (127), and rabbit muscle enzyme (68). An E m at 280 nm = 9.13 has been reported for the rabbit muscle enzyme (133). The atypical absorption spectrum with a maximum at 275-276 nm observed by Lee (132) is indicative of contaminating bound nucleotides. 

Subunit B1 of . coli ribonucleotide reductase is an SH protein carrying several binding sites of different affinity for substrate and effector nucleotides these interactions will be discussed in a later paragraph. The most unusual features of the smaller subunit B 2 are its iron content and a tyrosine residue present as stable free radical. Both these components are essential for enzyme catalysis and structurally coupled. They confer characteristic light absorption to the protein, with a broad maximum around 370 nm (a =... 

In order to make quantitative measurements. Miles (1958f)) has determined integrated infrared absorption intensities for some nucleosides, nucleotides, and polynucleotides in DjO solution (Fig. 12.8 and Table 12.2) by application of Ramsay s method I (Ramsay, 1952). When there is a well-resolved band, the application of Ramsay s method encounters no difficulty, but when overlapping bands occur, as in uridine and its derivatives, some uncertainty exists in determining the halfband width of a particular band. (The half-band width is a factor in Ramsay s equation and is defined as, Av, 2 = the width of the band in cm" at half-maximum intensity.) The equation as used by Miles to obtain A, the true integrated absorption intensity... 

Generally, UV absorption spectra can be utilized to determine the family to which an unknown base, nucleoside, or nucleotide belongs. Indeed, the chro-mophore of the nucleosides or nucleotides is typical of the purine or pyrimidine structvu-e, with different maximum absorption wavelengths in the adenosine, guanosine, cytosine, or thymidine series. For example, it is possible to differentiate readily the UV absorption spectra of guanosine (G), G-monophosphate (GMP), deoxy GMP (dGMP), G-diphosphate (GDP), and G-triphosphate (GTP), from those of adenosine (A), AMP, dAMP, ADR, and ATP. 

A potassium bromide pellet prepared from the major component indicated the formation of an ester linkage, the presence of a phosphate, and an A -t-butoxycarbonyl group. A UV spectrum with a maximum absorption at 257.5 nm identical to that of ATP eliminated the possibility that aminoacylation may have taken place at 6-amino group of nucleotide. Nuclear magnetic resonance spectroscopy demonstrated the presence of tertiary butyl protons. On the basis of the above evidence, structure (II) has been assigned to this product. 

Fig. 22. Ultraviolet absorption of pyridine nucleotides. There is a maximum at 340 m/i for the reduced form.

---