Elucidating the Role of Optical Activity of Polymers in Protein-Polymer Interactions

Abstract

Proteins are biomolecules with potential applications in agriculture, food sciences, pharmaceutics, biotechnology, and drug delivery. Interactions of hydrophilic and biocompatible polymers with proteins may impart proteolytic stability, improving the therapeutic effects of biomolecules and also acting as excipients for the prolonged storage of proteins under harsh conditions. The interactions of hydrophilic and stealth polymers such as poly(ethylene glycol), poly(trehalose), and zwitterionic polymers with various proteins are well studied. This study evaluates the molecular interactions of hydrophilic and optically active poly(vitamin B5 analogous methacrylamide) (poly(B5AMA)) with model proteins by fluorescence spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and circular dichroism (CD) spectroscopy analysis. The optically active hydrophilic polymers prepared using chiral monomers of R-(+)- and S-(-)-B5AMA by the photo-iniferter reversible addition fragmentation chain transfer (RAFT) polymerization showed concentration-dependent weak interactions of the polymers with bovine serum albumin and lysozyme proteins. Poly(B5AMA) also exhibited a concentration-dependent protein stabilizing effect at elevated temperatures, and no effect of the stereoisomers of polymers on protein thermal stability was observed. NMR analysis, however, showed poly(B5AMA) stereoisomer-dependent changes in the secondary structure of proteins.

Keywords: antifouling; chiral materials; protein stabilizing; protein–polymer interactions.

Figure 1

Study of Trp fluorescence intensity of BSA (concentration: 32 µg/mL) upon adding different w/w ratios (0, 0.5, 1, 2, 4, and 8) of polymer/protein in PBS of pH 7.4 for (A) poly(R-(+)-B5AMA₃₈), (B) poly(S-(+)-B5AMA₃₈), (C) poly(R/S-(+)-B5AMA₃₈), and (D) PEG at various time points (0, 2, 4, 6, 8, and 24 h). The average values are presented and the error bars represent the standard deviation (SD) for three individual measurements. “0” represents pure BSA solution in the experiments.

Figure 2

Study of Trp fluorescence intensity of LYZ (concentration: 32 µg/mL) upon adding different mass ratios (0, 0.5, 1, 2, 4, and 8) of polymer/protein in PBS of pH 7.4 for (A) poly(R-(+)-B5AMA₃₈), (B) poly(S-(+)-B5AMA₃₈), (C) poly(R/S-(+)-B5AMA₃₈), and (D) PEG at various time points (0, 2, 4, 6, 8, and 24 h). The average values are presented and the error bars represent the standard deviation (SD) for three individual measurements. “0” represents pure LYZ solution in the experiments.

Figure 3

Normalized fluorescence intensity of BSA with and without (A) poly(R-(+)-B5AMA₃₈), (B) poly(S-(+)-B5AMA₃₈), (C) poly(R/S-(+)-B5AMA₃₈), and (D) PEG. Normalized fluorescence intensity was determined at polymer/protein mass ratios of 1 and 8 at different time points. The average values are presented and the error bars represent the standard deviation (SD) for three individual measurements.

Figure 4

The characterization of polymer–protein interactions using ANS fluorescence dye upon changes in polymer/BSA (32 μg/mL) mass ratios (0, 0.5, 1, 2) for (A) poly(R-(+)-B5AMA₃₈), (B) poly(S-(+)-B5AMA₃₈), (C) poly(R/S-(+)-B5AMA₃₈), and (D) PEG. The average values are presented and the error bars represent the standard deviation (SD) for three individual measurements. “0” represents pure BSA solution in the experiments.

Figure 5

CD spectra indicating the interactions between indicated polymer–BSA mixtures prepared at a w/w ratio of 1. The spectrum of BSA alone is overlaid with different spectra representative of BSA conformation in a given condition derived by the subtraction of the CD spectrum of polymer alone from that of the polymer–BSA mixture.

Figure 6

Thermal denaturation study of BSA in the presence of different polymers. Normalized CD signal at 222 nm observed as a function of temperature during thermal denaturation of BSA for polymer/BSA w/w ratios of 1 (A) and 8 (B). Lines of fit using the logistic sigmoidal model (Equation (1)) are shown for all datasets except poly(R/S-(+/−)B5AMA)/BSA prepared at a w/w ratio of 8 due to poor reliability of that fit. The change in ellipticity at 222 nm between the minimum (low temperature) and maximum (high temperature) observed for the indicated sample during thermal denaturation was monitored by CD spectroscopy (C). Each sample was prepared in PBS at pH 7.4 with a final concentration of 0.5 mg/mL BSA. Normalization is presented with respect to extremes for a given sample type, not with respect to global extremes in ellipticity.

Figure 7

Titration of BSA in D₂O ¹H NMR signals upon incubation with (A) poly (R-(+)-B5AMA₃₈), (B) poly(S-(−)-B5AMA₃₈), (C) poly(R/S-(+/−)-B5AMA₃₈), and (D) PEG; in order from bottom to top are polymer alone (red), protein alone (purple), and polymer/protein w/w ratio of 1 (blue) and 2 (green).