Enzymic activity of chymotrypsin and its poly(ethylene glycol) conjugates toward low and high molecular weight substrates

Abstract

Native chymotrypsin and its polyethylene glycol (PEG) conjugates, obtained using the succinimidyl carbonate of methoxy-PEG (SC-PEG) as the amino group modifying reagent, were tested for their activity toward several low and high molecular weight substrates. Tripeptide and tetrapeptide p-nitroanilides either as N alpha-benzyloxycarbonyl derivatives or attached via their N-terminals to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers or to PEG, as well as the proteins albumin and azoalbumin, were used as substrates. Extensively modified chymotrypsin bearing on average of 14 PEG chains per chymotrypsin molecule (PEG14CHT) and a moderately modified one containing 10 PEG chains (PEG10CHT) both degraded the low molecular weight p-nitroanilides at rates comparable to, and in the case of PEG10CHT, greater than, the rates exhibited by the native enzyme. Synthetic high molecular weight substrates and azoalbumin were also degraded by the PEG-modified enzymes. However, rates of such enzymatic reactions were lower for the PEG-enzymes than for the native one. Native albumin, as compared to azoalbumin, resisted degradation by both PEGCHTs yet was readily digested by the native chymotrypsin. The results obtained indicate that substrate-size-dependent specificity of PEG-modified enzymes cannot be explained solely by steric hindrance considerations. The decreased activity of PEG-enzymes toward protein substrates is consistent with the well-documented ability of PEG to exclude proteins from its surroundings and with the influence of protein unfolding on the susceptibility to degradation.

Bioengineering functional copolymers. III. Synthesis of biocompatible poly[(N‐isopropylacrylamide‐co‐maleic anhydride)‐g‐poly(ethylene oxide)]/poly(ethylene imine) macrocomplexes and their thermostabilization effect on the activity of the enzyme penicillin G acylase

“…It is a well‐known fact that water‐soluble polymers, including poly(ethylene glycol), have a stabilization effect after conjugation to an enzyme (trypsin and chymotrypsin) through the introduction of free and mobile functional macromolecular groups 38, 39. Adding substances such as poly(ethylene glycol) does not always have a stabilization effect.…”

Synthesis of Temperature/pH-Sensitive Hydrogels Containing Disulfide Linkages as Cross-Links and Their Characterization

“…Absolute content (mol g- 1 ) of MAGlyGlyONp in copolymers was determined spectrometrically in DMSO (274 nm, £=9600 M-1 cm-1 ). 18 Owing to possible occurrence of ionization from AAc comonomer in DMSO-d 6 that might lead to mis-calculation of AAc content from NMR characterization, the copolymers (100 mg each) were subjected to aminolysis with 3-aminopropanol and content of AAc was determined by titration (Mettler DL-53) with 0.01 M NaOH. Average molecular weights of copolymers were Cloud points of copolymers were evaluated by measuring temperatures at which 10% reduction in transmittance at 500 nm from copolymer aqueous solutions (2.0 mg mL -1; phosphate buffer, pH 7.4) was observed spectrometrically.…”

Synthesis of semitelechelic poly[N-(2-hydroxypropyl)methacryl-amide] by radical polymerization in the presence of alkyl mercaptans

“…Modification of therapeutic proteins or biomedical surfaces with synthetic polymers can be used to favorably modify their properties [1][2][3][4][5] . Due to the multifunctionality of proteins or surfaces, semitelechelic macromolecules possessing only one functional group per polymer chain have been the modifiers of choice.…”