Nucleic acid absorption spectra

Spectral data (Amax and AE, and e x 10 3) for the pyrimidine bases investigated in a few representative papers are collected in Table XXVIII. The absorption bands are denoted by the capital letters A, B, C, etc. In Table XXVIII we have listed the results of the vacuum ultraviolet measurements by Yamada and Fukutome428 (cf. also ref. 429), who measured the spectra of sublimed films of cytosine, thymine, uracil (and also of guanine and adenine) down to 120 nm at room temperature. Several remarkable absorption peaks were found below 190 nm in addition to the already known ones near 260 and 200 nm. A weak absorption at 230-240 nm in cytosine was not indicated in the sublimed films of the molecule,428 but was visible in the stretched polyvinyl alcohol film spectrum.432 Crewe et al.i3° studied the interactions of fast electrons with the five nucleic acid bases and measured the energy-loss spectra of 20 keV electrons transmitted through thin films of these bases. These last data are also listed in Table XXVIII for comparison with the other spectral findings. 

Direct labeling of a biomolecule involves the introduction of a covalently linked fluorophore in the nucleic acid sequence or in the amino acid sequence of a protein or antibody. Fluorescein, rhodamine derivatives, the Alexa, and BODIPY dyes (Molecular Probes [92]) as well as the cyanine dyes (Amersham Biosciences [134]) are widely used labels. These probe families show different absorption and emission wavelengths and span the whole visible spectrum (e.g., Alexa Fluor dyes show UV excitation at 350 nm to far red excitation at 633 nm). Furthermore, for differential expression analysis, probe families with similar chemical structures but different spectroscopic properties are desirable, for example the cyanine dyes Cy3 and Cy5 (excitation at 548 and 646 nm, respectively). The design of fluorescent labels is still an active area of research, and various new dyes have been reported that differ in terms of decay times, wavelength, conjugatibility, and quantum yields before and after conjugation [135]. New ruthenium markers have been reported as well [136]. 

The content of long-chain PolyPs may be estimated by measuring the metachromatic effect in the absorption spectrum of toluidine blue (Chernysheva et al., 1971 Leitao et al., 1995 Lorenz and Schroder, 1999). Toluidine blue in an aqueous solution exhibits a concentration-dependent absorption spectrum due to a monomer (A.max, 632 nm)-dimer O.max, 590 nm) equilibrium. The PolyP induced the maximal shift of the absorption spectrum to 545 nm. Nucleic acids also induce metachromasia, but with a shift of about 570 nm with DNA and 590 nm with RNA. Figure 2.3 demonstrates the typical absorption spectra of toluidine blue and toluidine blue with different preparations of PolyPs (Chernysheva etal., 1971). 

This increase in the absorption spectrum following denaturation (destruction of secondary structure) is termed the hyperchromic effect (Fig. V-9). Conversely, the decrease in the absorption spectrum on renaturation of these types of nucleic acids (restoration of secondary structure) is termed the hypochromic effect. These effects are observed in Experiment 19. 

Fig. 2. Effect of native calf thymus DNA, denatured calf thymus DNA and yeast RNA on the visible absorption spectrum of tilorone in 0.01 M Tris-HCl (pH 7.0). Curve 1 is the spectrum of free tilorone (4.25 x 10-4 M). Other curves depict the spectra of tilorone in the presence of yeast RNA (curve 2), denatured DNA (curve 3) and native DNA (curve 4). Molar concentrations of nucleic acids (2 x 10 3 M) refer to phosphorous content of the polymer...

The absorption spectra of three yeast protein preparations prepared by different procedures were compared (Fig. 8). The presence of nucleic acid which has a X maximum at 260 nm tend to shift the absorption spectrum of yeast protein to lower wavelengths. The ratio of absorption at 280 to 260 nm is indicative of NA contamination in protein samples a ratio of more than one indicates pure protein devoid of nucleic acid whereas a ratio of 0.65 indicates approximately 30% contamination with NA. The yeast protein extracted with alkali and directly acid precipitated showed a X max at 260, a 280/260 ratio of 0.67 and contained 28%, NA determined chemically. Protein extracted in alkali, adjusted to pH 6 and incubated at 55°C for 3-5 hours, to reduce NA with endogenous ribonuclease, had a X max at 260, a 280/260 ratio of 0.8 and a NA content of 3.3% while yeast protein prepared by the succinylation procedure and precipitated at pH 4.5 showed a X max at 275 nm, a 280/260 ratio of 1.0 and nucleic acid content of 1.8. 

A nucleic acid gives more than 40 well-defined absorption bands in the IR spectrum (300-4000 cm region). For elucidating the nature of the normal vibrations of a nucleic acid, it is helpful to examine the effects of base composition on the vibrational frequencies, as well as the intensity and anisotropy of the IR absorption. Three of the four natural bases of a DNA (adenine, guanine, and cytosine) have an amino group w hich is considered to be nearly coplanar, because the purine or pyrimidine rings have aromatic character and the C—N bonds have some double-bond character. 

Nucleic acid also was found in association with the tuberculin protein. Spiegel-Adolf and Seibert investigated the problem spectrographically, and found that the tuberculin protein ( P.P.D. ) precipitated by trichloroacetic acid exhibited an absorption band at 2650-2670 A. This band was identical with that displayed by deoxyribonucleic acid. Precipitation of the tuberculin protein by ammonium sulfate afforded a product which did not display an absorption band in the ultraviolet region of the spectrum. The substance had a lower phosphorus content than that of products obtained by trichloroacetic acid precipitation. The biological activity was not impaired in any way. 

Many unusual nucleotides have been found as minor components of nucleic acids, especially in the soluble or transfer ribonucleic acids. Most of these minor components contain methylated aglycons in their structure. A review of these nucleotides has been presented by Dekker, and general techniques for their isolation as nucleosides have been reported by Hall. In addition, 5,6-dihydrouridylic acid (34) has been isolated by enzymic hydrolysis of certain transfer ribonucleic acids from yeast, and 4-thiouridylic acid (35) was obtained from the alkaline hydrolyzate of transfer ribonucleic acid from Escherichia coli. A nucleotide whose ultraviolet absorption spectrum was very similar to that of 2-thiouridine has been reported to be present in transfer ribonucleic acid. Although the a anomer (36) of cytidylic acid has been detected (and identified) in a yeast ribonucleic acid hydrolyzate, it is not certain whether this -cytidylic acid is a minor component of ribonucleic acid or an artifact produced during the alkaline hydrolysis. Among the minor nucleotide components of transfer ribonucleic acid, pseudouridylic acid (37)89-98 jg unique, in that the D-ribosyl moiety is linked to the aglycon... 

Reactions of neutral platinum complexes with nucleic acid components in vitro. Changes in the ultraviolet absorption spectrum of salmon sperm DNA after reaction with either cis or... 

Crystals from water + acetone, mp 196-200". [alff -47,5 (c = 2, 2% NaOH) -26.0 (c = 2, 10% HCI). pK, -- 3.8 pKj — 6.2. aM (molar absorbancy) 15.4 X I03 at 259 nm (pH 7.0). Readily sol in boiling water. The compound is readily deaminated by nitrous acid to form inosinic acid less rapidly hydrolyzed than 3 -adenylic acid by sulfuric acid. Furfural is formed only in traces On distillation with 20% HCI, cf. Levene, Bass, Nucleic Acids (New York, 1931) pp 230-232. Absorption spectrum Kalckar. foe. cir. thERap cat-. Nutrient. 

Absorption spectrum Voet et aL, Biopolymers 1, 193 (1963). Reviews see Guanine, Nucleic Acids. 

Spectra of proteins and nucleic acids. Most proteins have a strong light absorption band at 280 nm (35,700 cm ) which arises from the aromatic amino acids tryptophan, tyrosine, and phenylalanine (Fig. 3-14). The spectrum of phenylalanine resembles that of toluene (Fig. 23-7)whose 0-0 band comes at 37.32 x 10 cm. The vibrational structure of phenylalanine can be seen readily in the spectra of many proteins (e.g., see Fig. 23-llA). The spectrum of tyrosine is also similar (Fig. 3-13), but the 0-0 peak is shifted to a lower energy of 35,500 cm (in water). Progressions with spacings of 1200 and 800 cm are prominent. The low-energy band of tryptophan consists of two overlapping transitions and The Lb transition has well-resolved vibrational subbands, whereas those of the La transition are more diffuse. Tryptophan derivatives in hydrocarbon solvents show 0-0 bands for both of these transitions at approximately... 

Formation, decay, and absorption spectra of transient species produced by reaction of OH free radicals with DNA and nucleic acid constituents have been investigated by pulse radiolysis. Dilute aqueous N20 saturated solutions were exposed to 2-500 nsec, pulses (750-1700 rads/pulse) of 10 Mev. electrons, and changes in optical density were measured. The rate constant for formation of the DNA transient (nucleotide M.W. 350) is 6 X 108 M I seer1. The transient persists for more than 1 msec, with no significant change in spectrum. Thymine, uracil, deoxyribose, and thymidine transients have complex decay patterns which vary with pH. Comparisons of transient spectra confirm that the sites of attack by OH on thymine, 5-methylcytosine, and thymidine depend on pH. Abstraction from 5,6-dihydro-thymine is slower than addition to thymine. 

The pulse radiolysis technique has been used to measure absolute rate constants for reactions of some nucleic acid constituents with Clf radicals (the species produced by reaction of OH radicals with chloride ions in acid aqueous solution). The rate of disappearance of the Cl2 absorption spectrum was measured in the absence and presence of the various solutes. Rate constants for the corresponding OH radical reactions are found to be 20 to 200 times greater than the rate constants for the Clf radical reactions. Steady state radiolysis showed that in some cases the radicals produced by reaction of these compounds with Clf radicals differ in their subsequent reaction from the corresponding OH radical adduct. 

Because the OH absorption spectrum is in the ultraviolet region of the spectrum (28), where all nucleic acid derivatives absorb strongly, OH reaction rates in these experiments were measured using a modification of the competition method of Adams et al. (1, 2,3, 4, 5). In this method, the competitive solute (CNS-) forms a long lived species (P) absorbing at 500 m/x following its reaction with -OH at a rate k2. The solute (S) competes with CNS" for OH at a rate k.0H reducing the yield of P (Reactions 1 and 2). 

---