The structure and hydrogen bonding of water in the vicinity of carboxybetaine homopolymer (poly[1-carboxy-N,N-dimethyl-N-(2'-methacryloyloxyethyl)methanaminium inner salt] (PolyCMB), and a random copolymer of CMB and n-butyl methacrylate, Poly(CMB-r-BMA), with various molecular weights were analyzed in their aqueous solutions and thin film with contours of O-H stretching of Raman and attenuated total reflection infrared (ATR-IR) spectra, respectively. The relative intensity of the collective band (C value) corresponding to a long-range coupling of O-H stretchings of the Raman spectra for aqueous solution of Poly(CMB-r-BMA) was very close to that for pure water, which is in contrast with the smaller C value in aqueous solution of ordinary polyelectrolytes. The number of hydrogen bonds collapsed by the presence of one monomer residue (N(corr) value) of PolyCMB and Poly(CMB-r-BMA) (CMB, 45 mol %) (M(w), 1.14 x 10(4) and 1.78 x 10(4), respectively) could be calculated from the C value. The N(corr) values were much smaller than those for ordinary polyelectrolytes and close to those for nonionic water-soluble polymers such as poly(ethylene glycol) and poly(N-vinylpyrrolidone). Furthermore, a water-insoluble Poly(CMB-r-BMA) with a large BMA content (M(w) = 347 kD, CMB 27 mol %) could be cast as a thin film (thickness, ca. 10 microm) on a ZnSe crystal for the ATR-IR analyses. At an early stage of sorption of water into the Poly(CMB-r-BMA) film, the O-H stretching band of IR spectra for the water incorporated in the film was similar to that for free water, which is in contrast with the drastic change in the O-H stretching band of water incorporated in polymer films such as poly(methyl methacrylate) (PMMA) and poly(n-butyl methacrylate) (PBMA). The theoretical vibrational frequency for water molecules hydrating a betaine molecule calculated by using a density functional method supported the experimental results. The adhesion of human platelets to Poly(CMB-r-BMA) films was much less than that to PMMA and PBMA. With an increase in the content of CMB residue, the number of platelets adhered to the Poly(CMB-r-BMA) film drastically decreased and then gradually increased, probably due to the increase in the roughness of the film surface. These results suggest that the carboxybetaine monomer residues with a zwitterionic structure do not significantly disturb the hydrogen bonding between water molecules in both aqueous solution and thin film systems, resulting in the excellent blood-compatibility of the carboxybetaine polymers.
The state of sorbed water and the sorbing processes of water to various polymer thin films were studied with Fourier transform infrared (FTIR) spectroscopy. To prepare the polymer films, we used poly(ethylene glycol)s of different molecular weights and various kinds of vinyl polymers, such as poly(2‐methoxyethyl acrylate). The OH stretching band of water sorbed in the films increased gradually on contact with water vapor at 50% relative humidity and leveled off. When OH stretching bands of water sorbed to polymer films were compared, the peak positions and profiles of water sorbed to the polymeric materials with the same hydrogen‐bonding site were similar. A hybrid density‐functional method supported the assignment of the peaks. Furthermore, the diffusion coefficient (D) of water vapor in the polymer films was estimated by time‐resolved measurements of the sorbed water at the very initial stage (0–830 s). It was clearly shown that the D values of water vapor in the polymer materials with a strong hydrogen‐bonding site were smaller than those in hydrophobic polymers. The usefulness of the FTIR technique to investigate water sorption to polymer materials was definitely demonstrated. © 2001 John Wiley & Sons, Inc. J Polym Sci Part B: Polym Phys 39: 2175–2182, 2001
A comparison of the pressure and temperature dependences of the catalytic reaction of thermolysin, a thermostable neutral protease from Bacillus thermoproteolyticus, with those of a non-thermostable neutral protease from Bacillus subtilis revealed a distinct difference in K, values of these enzymes for 3-(2-furyl)acryloyl-blocked dipeptide and tripeptide substrates, but not for k,,,. Namely, the volume changes for the binding process ( A v) for these substrates and several competitive inhibitors were -20 --30 mljmol for thermolysin and nearly 0 mljmol for the non-thermostable neutral protease. The enthalpy and entropy changes for the binding process were negative for thermolysin, but positive for the latter enzyme. The activation volumes (LIP'') for the k,,, process were 25 -35 ml/mol for both proleases, and activation enthalpy and entropy showed no significant difference between the two enzymes. The characteristic difference in the pressure and the temperature dependences seen for the binding process is discussed in relation to the thermostability of the proteases.Thermolysin, a thermostable microbial neutral protease [I], is one of the representative zinc-containing proteases [2] and has attracted keen attention from many researchers. Zinccontaining proteases are widely distributed and some of them play very important roles in regulations and processings [3]. Recently, some microbial zinc-containing neutral proteases have been utilized to produce several functional polypeptides by a condensation reaction [4,5]. We have so far studied the reaction mechanisms of several types of proteases and in the study we used the pressure-dependence measurement of enzyme reactions as a powerful tool [6,7]. Just like temperature, the pressure affects chemical reactions drastically and the investigation of the pressure dependence has contributed considerably to the elucidation of the reaction mechanisms, even with reactions in solution. Enzyme reactions are not exceptional 181. In the previous paper, we investigated the detailed pH and temperature dependences of the thermolysin reaction and tried to assign the catalytic groups and proposed the mechanisms of the catalytic process involving the zinccoordinating water molecule [9]. In the present paper, we extend the investigation to the measurement of pressure dependence and compare the results with those of a neutral protease from Bacillus subtilis, a non-thermostable control of thermolysin, as well as with the results previously obtained with carboxypeptidase A [7], to understand further the reaction mechanisms of this group of enzymes.Abbreoiations. FuaGly-PheNH,, 3-(2-furyl)acryloylglycyl-~-phenylalaninaniide ; FuaGly-LeuNH, ,3-(%-furyl)acryloylglycyl-~-leucinamide; FuaGly-Phe-Ala, 3-(2-furyl)acryloylglycyl-~-phenylalanyl-~-alanine; FuaGly-Leu-Ala, 3-(2-furyl)acryloylglycyl-~-leucyl-~-alanine; ZPhe, carbobenzyloxy-rA-phenylalanine; ZGly-Leu, carbobenzyloxyglycyl-L-leucine ; Hepes, 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid; Mes, 4-morpholineethanesulfonic acid; Me,S...
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