Hypercoiling

Finally the results recently reported by DUBIN and Strauss are certainly worth mentioning, Potentiometric titration of alternating copolymers of maleic anhydride and n-butyl ether indicates that there occurs a conformational transition which is not exhibited by a copolymer of maleic anhydride and ethyl vinyl ether (Fig. 7). The anomalous behavior of the butyl copol3mier is clearly cormected with the establishment of hydrophobic interactions among the butyl groups. Dubin and Strauss conclude that the maleic anhydride and n-butyl vinyl ether copolymer is hypercoiled in dilute aqueous solution. These authors also point out that the behavior of the latter copolymer is identical with that previously found for highly charged polysoaps. 

Dubin, P., and U. P. Strauss Hydrophobic hypercoiling in copolymers of maleic add and alkyl vinyl ethers. J. Phys. Chem. 71, 2757 (1967). 

Time-resolved fluorescence spectroscopy and fluorescence anisotropy measurements have been applied to study (i) excimer formation and energy transfer in solutions of poly(acenaphthalene) (PACE) and poly(2-naphthyl methacrylate) (P2NMA) and (ii) the conformational dynamics of poly(methacrylic acid) (PMA) and poly (acrylic acid) as a function of solution pH. For PACE and P2NMA, analysis of projections in which the spectral, temporal and intensity information are simultaneously displayed have been used to re-examine kinetic models proposed to account for the complex fluorescence decay behaviour that is observed. Time-resolved fluorescence anisotropy measuranents of fluorescent probes incorporated in PMA have led to the proposal of a "connected cluster" model for the hypercoiled conformation of this polymer existing at low pH. 

A further application of time-resolved fluorescence measurements is in the study of conformational dynamics of polymer chains in solution. Fluorescence anisotropy measurements of macromolecules incorporating suitable fluorescent probes can give details of chain mobility and polymer conformation (2,14). A particular example studied in this laboratory is the conformational changes which occur in aqueous solutions of polyelectrolytes as the solution pH is varied (15,16). Poly(methacrylic acid) (PMA) is known to exist in a compact hypercoiled conformation at low pH but undergoes a transition to a more extended conformation at a degree of neutralization (a) of 0.2 to 0.3 (1 6). Similar conformational transitions are known to occur in biopolymer systems and consequently there is considerable interest in understanding the nature of the structures present in model synthetic polyelectrolyte solutions. 

One explanation that can be offered to explain the two Tc values obtained for PMA at low values of a is that they represent the independent rotation of small clusters of the polymer chain. The larger value of (approximately 50 ns) can be associated with a rotating spherical cluster of radius 3.8 nm and of polymer molecular weight equal to 19000. Rotating units of similar size have been observed when the probes 9-methylanthracene and 9,10-DMA are solubilized in the PMA hypercoil structure (15,16) and when the more polar fluorescent probes Rhodamine B ( ) and 1,8-anilinonaphthalene sulfonic acid (1,8-ANS) (28) are bound to PMA for a value of a equal to 0. The smaller rotating unit present in PMA and PAA whose value of Tj, is approximately equal to 5 ns (which corresponds to particles whose radii are approximately equal to 2 nm) may arise from the rotation of a small section of the chain which is just sufficient to surround the 9,10-DMA probe and protect it from unfavourable entropic interactions with water. This shorter T, was... 

In later work it was shown that water-soluble antennas could be made by copolymerizing aa oinatic monomers such as vinyl naphthalene and naphthylmethyl methacrylate with polyelectrolytes such as acrylic acid [3,4]. The high efficiency of these antennas in dilute aqueous base was attributed to the hypercoiling of the poly-(acrylic acid) chain to give a pseudo micellar stincture such as that illustrated schematically in Figure 2. We believe that such structures are formed spontaneously in solution due to the hydro-phobic interactions of the large aromatic ccmiponents stabilized by the interaction of water with the hydrophillic carboxyl anions. 

It was later shown that other types of polyelectrolytes involving partial sulfonation of poly(vinylnai ithalene) [5,6] and copolymers of aromatic monomers with styrene sulfonate would also lead to polymers which in aqueous solution achieved this hypercoiled configuration. 

Figure 2. Proposed hypercoiled structure of anthracene end-trapped copolymers of auirylic acid and NMMA in dilute alkaline solution lowing Fbrster radii for various energy-transfer processes. ...

For example, simple fluorescence intensity measurements on dispersed hydrocarbon probes such as anthracene [17], perylene [18], pyrene [6,17,22], 9,10-dimethylanthracene (DMA) [60], and coumarin dyes [62] have confirmed that PMAA displays pH-dependent solution behavior. A marked decrease in the intensity of the probe occurs between pH 5 and 6, which coincides with the conformational transition of PMAA as determined by classical methods [2-4,47-50]. Two interrelated effects account for this behavior the solubilizing capacity of the polymer promotes an increase in the concentration of the probe in the solution [6,17,18,60,62] and because the intensity of the fluorescence observed is proportional to the excited state population the resulting emission is enhanced. The hydrocarbons may also be considered to be preferentially solubilized within the hydrophobic domains or structures of the hypercoiled state [6,22]. This results in a degree of protection from the deactivating effects of the aqueous phase and a concomitant increase in the fluorescence observed [6,17,18,22,60,62]. 

Water-soluble probes have also been reported to undergo marked changes in their fluorescence characteristics when dispersed in polyelectrolyte solutions [18,52,61,72-78]. For example, the cationic dye, auramine O (AuO) is virtually nonfluorescent in aqueous solution but an increase in intensity is observed in the presence of PMAA at low pH as a result of enhanced binding in the hypercoiled state [72,73,75,78]. On neutralization, the fluorescence from AuO decreases as the compact structure breaks down forming the expanded state and the probe is released to the aqueous phase [52]. In a recent extension of this theme, the sensitivity of the emission spectrum of AuO to the environment in which it resides has been further exploited by covalently bonding the dye to PMAA [61]. Figure 2.1 shows the... 

Comparison of the transient fluorescences at pH 5.7 (Fig. 2.2) and pH 3.2 (Fig. 2.3) reveals that the duration of the decay has increased at low degrees of ionization. This is consistent with solubilization of pyrene in the protective domains of the hypercoiled conformation. 

Examination of the kq values listed in Table 2.2 clearly reveals the conformational transition of the poly electrolyte for example, PMAA has been labeled with 1-pyrene acrylic acid (PyAA) [22], ACE [12], 1VN [12], and vinyl diphenylanthracene (VyDPA) [94] and quenched with nitromethane in aqueous media. The kq values derived from lifetime data for each of these labels are of the order of 0.5 x 109 moE1 dm3 s 1 at low degrees of ionization. The formation of the hypercoiled conformation provides a degree of protection for the excited state from the quencher, and a low value of kq results. 

A dramatic reduction in the quenching efficiency is apparent from the labeled PMAA samples (see Table 2.2) on use of / at a low pH. This reflects the fact that the anionic quencher is inhibited from accessing the hydrophobic hypercoil. At pH values in excess of 6, although PMAA is in an extended conformation, repulsion occurs between the I and the carboxylate ions which results in reduced kq values (ca. 0.2-0.05 x 109mol 1dm3 s 1) compared to that observed while using CH3NO2 under similar pH conditions (see Table 2.2). 

The polarization of a DMA-labeled PMAA sample was monitored [18] as afunction of pH, and rc was later derived [46] at various degrees of ionization, via Equation 2.28. tc varies from ca. 32 ns at a = 0 to ca. 6 ns at a = 0.8. Not surprisingly, the authors [46] offered a similar explanation for the pH dependence of xc to that of Anufrieva and Gotlib [16] essentially, abreakdown in the hypercoil structure occurs as a increases and the polyelectrolyte expands allowing increased mobility of the chain segments. 

When TRAMs are made on dispersed probes, tc will reflect the speed of rotation of the fluorophore [20,46,60,76], which can be related to the microviscosity of the medium. In the context of probing the structure of PMAA, the fluorescent dyes have been occluded in the hypercoiled conformation [46,60,76] allowing an estimate of the size of the rotating, solubilizing cluster to be derived from the resultant tc. Alternatively, if the fluorophore is covalently attached to the polyelectrolyte in the form of a label then, depending on its mode of attachment, information concerning motion of the chain ends [46,60,76], the backbone [26,88,112,113], and chain substituents [26,88] can be derived from tc. 

Early time-resolved anisotropy experiments on PMAA used a combination of anthryl-based labels and probes [18,46,60,76] in an effort to fully characterize the conformational switch of the polyelectrolyte in aqueous solution. In their study of probes dispersed in and labels incorporated into PMAA, Treloar and coworkers [60,76] derived information from anisotropy experiments pertinent not only to the cluster size of the rotating units, but also to the structure of the hypercoil itself. 

For the probes 9-methyl anthracene (MA) and DMA solubilized in the hypercoiled conformation of PMAA, the anisotropy decays could best be described by invoking double-exponential analysis of the form described by Equation 2.32... 

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