Nucleic acid hypochromicity

Kinoshita, Imoto etal.11 14) synthesized other anionic models, 5 (APVP), CPVP, UPVP, TPVA, HPVA, THPVA, and 6 (AMPPVA), by the polymer reaction of N-eoupled(2-dihydrogenphosphate)-ethylderivatives of nucleic acid bases (or adenosine-5 -phosphate, AMP) with polyvinylaleohol. A, C, U, T, H, and TH denote adenine, cytosine, uracil, thymin, hypoxanthine, and theophylline, respectively. The authors reported the apparent hypochromities of 3 to 16% for many kinds of mixtures of the models and DNA or RNA, as compiled in Table 1. However, for the mixtures APVA + RNA, HPVA + RNA HPVA + DNA, THPVA + RNA, CPVA + DNA and CPVA + RNA, no hypochromicity was detected. 

Table 1. Apparent hypochromicities of anionic models of nucleic acid...

It can be seen from the figure that the electrostatic repulsive forces between the macrocations are overwhelmed, probably by hydrophobic attractive forces between their hydrophobic side groups. It should be noted that the complimentary base-base pairing is unimportant in the present case. If this is not the case, the mixtures of APVP and TPVP should show the largest hypochromicity. This, however, is not the case. The importance of the hydrophobic interactions between nucleic acid bases has been proposed by Ts o et al.I9 from thermodynamic parameters of various nucleic acid bases or nucleosides in aqueous media. 

A hypochromicity was observed between THPVP and APVP (or TPVP). Since theophylline is not a nucleic acid base and does not form hydrogen-bonding, these observations indicate that stacking-type hydrophobic forces are important. 

Poly A form a complex with a 4 1 stoichiometry. The apparent hypochromicities of various mixtures are listed in Table 4. The mixtures of A12 with Poly U and of T12 with Poly A showed large hypochromicities compared with other mixtures, which suggests the importance of the hydrogen-bonding formation between complementary nucleic acid bases such as A-U and T-A. 

The close interaction between stacked bases in a nucleic acid has the effect of decreasing its absorption of UV light relative to that of a solution with the same concentration of free nucleotides, and the absorption is decreased further when two complementary nucleic acids strands are paired. This is called the hypochromic effect. Denaturation of a double-stranded nucleic acid produces the opposite result an increase in absorption... 

A plot of the optical absorbance at 260 nm (the wavelength of maximum light absorption by nucleic acids) versus temperature is known as a melting curve (Fig. 5-45). The absorbance is lower, by up to 40%, for native than for denatured nucleic acids. This hypochromic effect (Chapter 23) is a result of the interaction between the closely stacked bases in the helices of the native molecules. The melting temperature Tm is taken as the midpoint of the increase in absorbance (Fig. 5-45). As the percentage of G + C increases, the nucleic acid becomes more stable toward denaturation because of the three hydrogen bonds in each GC pair. Tm increases almost linearly with increases in the G + C content. In the "standard" citrate buffer (0.15 M NaCl + 0.015 M sodium citrate, pH 7.0) Eq. 5-22 holds. The exact numerical relationship depends strongly upon the ionic composition and pH of the medium.37 72 552 553... 

Whereas proteins have their low energy absorption band at 280 nm, polynucleotides typically have maxima at 260 nm (38,500 cm ). A phenomenon of particular importance in the study of nucleic acids is the hypochromic effect. In a denatured polynucleotide the absorption is approximately the sum of that of the individual components. However, when a double helical structure is formed and the bases are stacked together, there is as much as a 34% depression in the absorbance at 260 nm. This provides the basis for optical measurement of DNA melting curves (Fig. 5-45).45,86 The physical basis for the hypochromic effect is found in dipole-dipole interactions between the closely stacked base pairs.7,86,87... 

Melting and helix formation of nucleic acids are often detected by the absorbance of ultraviolet light. This process can be understood in the following way The stacked bases shield each other from light. As a result, the absorbance of UV light whose wavelength is 260 nanometers (the Amo) of a double-helical DNA is less than that of the same DNA, whose strands are separated (the random coil). This effect is called the hypochromicity (less-color) of the double-helical DNA. 

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. 

Thus far, only one report of the UV resonance Raman excitation profiles of nucleic acids has appeared in the literature. The excitation profiles of calf thymus DNA [177] shows the same hypochromism as that observed in both single-stranded and duplex oligonucleotides. Also as expected, the excitation profiles are quite complex. Although an excitation profile is obtained for every vibrational mode, numerous bases are contributing to the Raman intensity observed in every vibration, each in its own microenvironment. Thus, the resonance Raman intensities currently are not useful for elucidating the excited-state structural dynamics of nucleic acids. 

In vitamin Be-deflcient experimental animals, there are skin lesions (e.g., acrodynia in the rat) and fissures or ulceration at the corners of the mouth and over the tongue, as well as a number of endocrine abnormalities defects in the metabolism of tryptophan (Section 9.5.4), methionine (Section 9.5.5), and other amino acids hypochromic microcytic anemia (the first step of heme biosynthesis is pyridoxal phosphate dependent) changes in leukocyte count and activity a tendency to epileptiform convulsions and peripheral nervous system damage resulting in ataxia and sensory neuropathy. There is also impairment of immune responses, as a result of reduced activity of serine hydroxymethyltransferase and hence reduced availability of one-carbon substituted folate for nucleic acid synthesis (Section 10.3.3). It has been suggested... 

From various physical and biophysical properties of nucleic acid analogs the most important property for the present purpose is their interaction with nucleic acids. The spectrophotometric methods for detection of complex formation were applied to all combinations of polyvinyl polynucleotide analogs and natural polynucleotides (Fig. 3). In aqueous media hypochromic complexes were formed in combinations where the bases in the polynucleotide and analog were complementary. Poly-l-vinylcytosine is soluble in aqueous-propylene glycol base-pair type complexes were detected there also. An analog of polyinosinate, poly-9-vinylhypoxanthine, is soluble only in solutions of a detergent, sodium dodecylsulfate. This detergent intercalates into the polymer and conveys to it an... 

Stacked bases in nucleic acids absorb less ultraviolet light than do unstacked bases, an effect called hypochromism. Thus, the melting of nucleic acids is easily followed by monitoring their absorption of light, which peaks at a wavelength of 260 nm (Figure 517). 

Because of their aromatic structures, the purine and pyrimidine bases absorb UV light. At pH 7 this absorption is especially strong at 260 nm. However, when the nitrogenous bases are incorporated into polynucleotide sequences, various noncovalent forces promote close interactions between them. This decreases their absorption of UV light. This hypochromic effect is an invaluable aid in studies involving nucleic acid. For example, absorption changes are routinely used to detect the disruption of the double-stranded structure of DNA or the hydrolytic cleavage of polynucleotide strands by enzymes. 

To estimate the concentration of an oligonucleotide solution, the absorption is measured at 260 nm. Correct weights of solid oligonucleotides are difficult to obtain due to non-stoichio-metric inclusion of water molecules into the solid. UV spectra of nucleic acids exhibit a pronounced hypochromicity. In double-stranded nucleic acids, base stacking with a distance of about 3.5 A lowers UV-absorption by up to 20% due ii-systems interaction when compared with single-stranded oligomers (Fig. 25). 

The absorption intensity of nucleic acids is lower than in mixtures of their component monomers. The hypochromicity exhibited by nucleic acids arises from the interaction between electronic states of nitrogen bases due to their constrained polymeric structures, i.e. 6p < XiEmi, where Cp is the extinction coefficient of nucleic acid, Xi and Em are the mole fraction and extinction coefficient of the ith monomer component. The percent hypochromicity at X (HO is defined as... 

For example, the temperature-induced transition between native and random coil states of a nucleic acid can be conveniently monitored by UV spectrophotometry. The reason for this is that stacked bases have a lower absorbance per base than unstacked bases this is called hypochromicity, which is defined as ... 

Hypochromic effect an optictil phenomenon in molecules with several chromophores in which the sum of the absorbance of the individual components is greater than the absorbance of the whole molecule. The absorbance of the nucleic acids at 260 nm, for example, is less than the calculated sum of the absorbances of the component bases The H.e. depends on the content of adenine and thymine and is therefore greater in DNA than in RNA. Double-stranded polynucleotides have a greater H. e. than single-stranded, because the effect is intensified by hydrogen bonds... 

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