Hypochromicity value

Figure 6 shows the clear interaction between atactic polyMAOA and atactic poly-MAOT in DMSO-ethylene glycol (3 2 v/v). The overall stoichiometry of the complex is 1 1 under the condition studied. The hypochromicity value obtained from Fig. 6, 14%, is greater than that obtained in trimethyl phosphate6. In DMSO, however, no interactions between these polymers were observed. 

Values were derived from the extinction coefficients of the 3 -monophosphate ribonucleotides (M. Alexis, Ph.D. Thesis, Univ. of London, 1978) at the wavelength maxima, absorption spectra of the 3 -monophosphates to convert peak values to those at 260 nm, and hypochromicity values for the RNA dimers (M. M. Warshaw, Ph.D. Thesis, Univ. of California, Berkeley, 1966). Monomer Ap, Cp, and Gp extinction coefficients and dimer hyperchromicity values were assumed to be the same for DNA and RNA the extinction coefficient for dTMP was taken to be the same as that for dT [C. R. Cantor, M. M. Warshaw, and H. Shapiro, Biopolymers 9, 1059 (1970)]. Values are for 0.1 M ionic strength, pH 7.0, 25°. 

Interaction of the synthetic polymers and polynucleotides were estimated from the hypochromicity values in UV spectra. The UV spectra were measured with a JASCO UV-660 spectrometer equipped with a temperature controller at 20 C. Polynucleotides were obtained from Yamasa Shoyu Co. Ltd. Water soluble poly(ethyleneimine) derivatives containing nucleic acid bases and polynucleotides were dissolved in Kolthoff buffer (pH 7.0, O.IM KH2PO4 -0.05M Na2B40 -10H20). These solutions were stocked for 2 days at 20 C, and then mixed to give a polymer mixture of 10 total concentration of nucleic acid base units in aqueous solution. 

For the system in question, time dependence of hypochromicity value was observed as shown in Figure 1. The hypochromicity was not observed in 3 hours after mixing of the polymer solutions (Figure la). The absorbance decreased (the hypochromicity increased) and then became constant after 3 days (Figure lb). The conformational change of the synthetic polymers should be thus important for the formation of a stable complex. 

Interactions between PEI-Hse-Hyp and poly(C) which contains the complimentary nucleic acid base can be clearly observed in aqueous solution at pH 7.0, as shown in Figure 3. The overall stoichiometry of the complex based on the nucleic acid base units was approximately 2 1 (hypoxanthine cytosine) under the conditions used. The maximum hypochromicity value (26%) was smaller than that of the poly (I) poly (C) (33%) system. 

Figure 4 shows the mixing curve between PEI-Hse-Cyt and poly(I) after 3 h and 3 days at pH 7.0 in the Kolthoff buffer aqueous solution. The stoichiometry of the complex was 2 1 (hypoxanthine cytosine) and the hypochromicity value was 7%. The stoichiometry of the PEI-Hse-Cyt poly(I) complex was similar to that of PEI-Hse-Hyp poly(C) system. 

The hypochromicity value was small as compared with that of the PEI-Hse-Hyp poly(C) system. Polynucleotides containing purine base form very stable structures by self association in aqueous solution. On the other hand, the structures of polynucleotides containing pyrimidine bases... 

Figure 5 shows the mixing curves for PEI-Hse-Ura with poly(A) at pH 7.0. From the curve in Figure 5b, the maximum hypochromicity value obtained was 54.4%. The overall stoichiometry of the complex based on nucleic acid base units was 1 1 (uracil adenine). 

As a control experiment, interaction between poly(A) and poly(U) was studied under the conditions used here. As shown in Figure 6, the complex formation was observed immediately after mixing of the polymer solutions, and for maximum hypochromicity value was 40.3%. The hypochromicity value for the PEI-Hse-Ura poly(A) system was higher than the poly(U) poly(A) system, PEI-Hse-Ade poly(U) system, and any other nucleic acid analog-polynucleotide system. 

Figure 8 shows the mixing curves of PEI-Hse-5FU (5FU) with poly(A) at pH 7.0. The maximum hypochromicity value was obtained for the base unit ratio of 2 1 (5-fluorouraciliadenine). The maximum hypochromicity value of the PEI-Hse-5FU poly(A) system was 40.3%, which was smaller than the PEI-Hse-Ura poly(A) system (54.4%), equal to the poly(U) poly(A) system (40.3%), and higher than the PEI-Hse-Thy poly(A) system (38.5%). These facts suggest that the incorporation of the 5-fluorouracil base results in a decrease in the stability of the polymer complex. 

Figure 9 shows the mixing curve between PEI-Hse-Ade (5a) and poly(U), for 3 hours (a) and 3 days (b) after mixing the polymer solution. In this case, the formation of the polymer complex was observed even after 3 hours, as shown in Figure 9a. The stoichiometry of the complex was 1 1 (thymine adenine) and the maximum hypochromicity value was 49.6% at this base ratio. This value was a little higher as compared with the values obtained for PEI-Hse-Thy PEI-Hse Ade and poly(A) poly (U) systems. The formation of the polymer complex, however, was negligible at pH 2.2, where the adenine base exists in a protonated form. From these facts, it should be concluded that formation of the polymer complex between PEI-Hse-Ade and poly(U) was caused by the specific interaction between adenine and uracil, and the self association of PEI-Hse-Ade in aqueous solution was negligible.

It is well known that the complimentary base of uracil is not cytosine but adenine. However, as seen in Figure 10b, the hypochromic effect was also observed for the interaction of PEI-Hse-Ura and poly(C), for 3 days after mixing the polymers. The overall stoichiometry of the system was 1 1 (uracilrcytosine). The maximum hypochromicity value (20.1%),... 

As a control experiment, the interaction between poly(U) and poly(C) was also studied under the conditions used. The complex formation was observed immediately after mixing the polymer solutions, and a similar curve was obtained even after 3 days. The maximum hypochromicity (5.5%) was observed at base unit ratio of 1 1 (uracilrcytosine). The maximum hypochromicity value of the poly(U)rpoly(C) system, however, was smaller than that for the PEI-Hse-Ura poly(C) system. Therefore, the interaction between PEI-Hse-Ura and poly(C) was concluded to be caused by the interaction between uracil and cytosine bases. 

The maximum hypochromicity values and stoichiometries of the polymer complexes are summarized in Table 2. In this table, the value of selectivity (S) was calculated by Equation 1, where H(a>, and H(c> are the maximum hypochromicity values of the uracil derivatives with poly(A) and with poly(C), respectively. The selectivity value means the selectivity of the uracil derivatives to the complementary poly(A) based on poly(C). PEI-Hse-Ura has the highest hypochromicity value for poly(A), but the selectivity value (s 2.7) is low. On the other hand, PEI-Hse-Thy has the lowest hypochromicity value for poly(A), but the selectivity value is... 

Table 2. The maximum hypochromicity values. Stoichiometry and the values of selectivity (S) for nucleic acid analogs - polynucleotides systems.

The data in Table 2, therefore, should indicate that the higher the hypochromicity values the lower the selectivity for the interaction with the complementary base. 

This trend was found to be caused by the nearest neighboring units, and to be independent of remote units. The hypochromicity values for the 1/1 mixture of adenine and thymine derivatives are tabulated in Table 1. The results suggest the affect of the molecular weight dependency of the intermolecular interaction. It can be seen from the Table that the interaction between the oligomers is fairly small, while interaction between the oligomers and the polymer are remarkable. The polymer effect observed for the intermolecular interaction may be caused by an entropy factor, because the polymer has a number of nucleic acid bases in one molecular chain as the interaction sites. 

In a physicochemical study of oligo- or polynucleotides, it has been reported that Hypochromicity, depends on the degree of polymerization [61]. The value of native DNA is about 60%, which corresponds to the Hypochromicity, of the absorption of its mononucleotides [60]. The present result indicates that the purine rings may intramolecularly stack by themselves in an aqueous solution... 

The existence of base stacking interaction for poly-VUr was also suggested from UV spectra25). At pH 12, the value of hypochromicity for poly-VUr was 29 to 51 % as compared to 1-ethyluradl. For poly-U solution, the value is only a few percent at room temperature while at lower temperature, about 30% of hypochromicity is observed which is attributed to the formation of a stacked helical polynucleotide structure. It seems therefore likely that the high value of hypochromicity observed for poly-VUr solution may be due to base stacking interactions. 

Fig. 7. Hypochromicity for the system of the l 1 mixture of isotactic polyMAOA-isotactic polyMAOU in a DMSO-EG mixture. Absorbance values were obtained in a 10 mm cell at 25 °C (O) after 1 day ( ) after 7 days...

It has been reported that the helicity of poly-L-lysine derivatives decreases with decreasing base content and the formation of complexes between polymers is affected by their helical content79. Effect of helicity of poly-L-lysine derivatives on the complex formation ability has also been observed (Fig. 27). The value of hypochromicity tends to decrease remarkably with falling thymine content in PLL-Ts. The overall stoichiometry of the complex of polyMAOA - PLL-T-65 was obtained as 5 1 (adenine thymine) it does not reflect the stoichiometry at the binding site (the theoretical stoichiometry of the binding sites for the polyMAOA-PLL-T-65 system is 3 2 (adenine thymine)). 

Aluminium overload in uremic patients can lead to a microcytic hypochromic anemia (45). The mechanism is unknown, but it appears to involve inhibition of heme synthesis, either by inhibition of enzyme activity or by interference with the incorporation or utilization of iron. Exposure for 3 months is sufficient to reduce hematological values and cause significant increases in serum and urinary aluminium (46). The anemia can be reversed by lowering the aluminium intake or by chelation therapy with deferoxamine. 

---