Abstract
Schizophyllans carrying poly(N-substituted-acrylamide)-branches were synthesized from native schizophyllan through Ce(IV)-initiated radical homo- or co-polymerization of N-iso-propyl- and N-sec-butyl-acrylamide. A series of structural analyses including elemental analysis, gel permeation chromatography, 1H NMR, and enzymatic degradation using β-1,3-glucanase revealed that schizophyllans (150 kDa) bearing long poly(N-substituted-acrylamide)-branches (ca. 290 kDa) are obtained. These SPG-conjugates form macromolecular complexes with certain homopolynucleotides such as poly(C), poly(dA), and poly(A) and then efficiently co-precipitate them on heating. For example, schizophyllan carrying poly(N-iso-propylacrylamide)-branches can selectively recover poly(C) from a mixture of poly(C) and poly(U) by simple heating (36 °C) followed by centrifugation. HPLC analysis of the recovered polynucleotide revealed the high poly(C)-selectivity (poly(C):poly(U) = 95:5). Similarly, schizophyllan carrying more hydrophobic poly(N-iso-propylacrylamide-co-N-sec-butylacrylamide)-branches precipitates at lower temperature (ca. 20 °C) and is applicable to isolation of poly(A) that forms less stable macromolecular complexes with SPG and SPG-derivatives (the dissociation temperatures are around 30 °C).
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Although one can expect that the copolymer consisting of one native SPG (150 kDa) and one poly(NIPAm) (290 kDa) should be 440 kDa, the observed Mw of SPG-NIP is only 320 kDa. We can exclude the possibility of SPG-degradation through radical polymerization step, since treating of SPG with Ce(IV) at 50 °C in the absence of NIPAm monomer did not result in a SPG-fragmentation (confirmed by GPC). We consider that this discrepancy comes from an unsuitable linear polysaccharide (pullulan) used as a calibration standard for the branched SPG derivatives.
Since SPG-NIP/poly(C) complex precipitates before the complex is dissociated, the precise Tm cannot be determined. We found, however, that when the precipitate is kept in aqueous 100 mM NaCl solution at 50 °C, the concentration of poly(C) in the supernatant increases gradually (monitored by UV–vis spectroscopy). On the other hand, when the same experiment was carried out at 40 °C, such an increase in the absorbance was not detected. The results imply that the Tm for SPG-NIP/poly(C) complex is lower than that of SPG/poly(C) complex and lies between 40 °C and 50 °C. Introduction of the poly(NIPAm)-branches should be attributable to the destabilization of the complex.
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SPG-coBP (20 mg mL−1, in DMSO, 100 μL) was mixed with poly(A) (1.0 mg mL−1, in water, 100 μL) and then, diluted with Tris–HCl buffer (12 mM, pH 8.0, [NaCl]=120 mM, 820 μL). The resultant solution was immediately subjected to centrifugation (24 °C, 100g, 20 min).
An incubation under 4 °C for 2 d is recommended, if the macromolecular complex with the highly ordered helical structure is required. However, such ordered complex is clearly not required for the quick precipitation procedure.
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Matsumoto, T., Umeda, M., Numata, M. et al. Poly(N-substituted-acrylamide)-branched Schizophyllans Are Useful for Selective Recovery of Homopolynucleotides through Convenient and Quick Precipitation Procedures. Polym J 37, 177–185 (2005). https://doi.org/10.1295/polymj.37.177
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DOI: https://doi.org/10.1295/polymj.37.177
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