Switching the enzymatic activity of ribonuclease a based on enzyme/polymer complex formation

Abstract

Context: Enzymes are ideal for various applications in both medicine and biotechnology. Switching on/off the enzymatic activity of enzymes by polymeric modification would expand the applications of enzymes in a wide range of research fields. Objectives: On/off switching the enzymatic activity of RNase A and the confirmation of the enzyme/polymer complex formation which leads to improve the heat resistance of RNase A. Materials and Methods: α-Methoxy-ω-methacryloyl poly (ethylene glycol) (PEG-MA) macromonomer and PEAMA-g-PEG were synthesized. Bovine RNase A, cytidine 2′,3′-cyclic monophosphate sodium salt (cCMP), and 3-(N-morpholino)propanesulfonic acid (MOPS) were obtained from Sigma Chemical Co. (St. Louis, USA). PAAc (Mn = 5,000 g/mol) were purchased from Wako (Osaka, Japan). Sodium dihydrogen phosphate dihydrate (NaH2PO4.2H2O) was obtained from Nacalai Tesque Inc. (Kyoto, Japan). The enzymatic activity of RNase A was estimated as follows. The RNase A concentration was determined by measuring the absorbance at 280 nm with an appropritate blank, using an extinction coefficient of 7.10 mL mg -1 cm -1 . A total of 1.5 mL of 0.1 mg/mL cCMP solution prepared in 0.1 M MOPS (pH 7.0) was mixed with 10 µL of the RNase A solution in 50 mM sodium phosphate buffer (pH 7.0). The increase in light scattering intensity of the solution was monitored by measuring the absorbance at 284 nm for 60 s in a UV-vis spectrophotometer at room temperature. After heat treatment, the enzymatic activities were measured. Far-UV and near-UV CD spectra were monitored using a spectropolarimeter (model J-720W; Jasco, Tokyo, Japan). Results: We have found that poly (acrylic acid) (PAAc) suppressed the enzymatic activity of RNase A completely and the recovery of enzymatic activity were observed (94%) by the external addition of PEAMA-g-PEG to the RNase A/PAAc complex. Our present findings suggest that the complexation between PEAMA-g-PEG and RNase A has occurred which improve the heat resistance (64%) of RNase A. Heat treatment has been carried out at 98°C for 10 minutes where the native RNase A lost all of its enzymatic activity. CD spectral analysis also indicates that the conformation of the enzyme was not altered due to the complexation. Conclusion: Poly (N,N-diethylaminoethyl methacrylate)-graft-poly (ethylene glycol) (PEAMA-g-PEG) restored the enzymatic activity of RNase A completely which was inactivated upon the addition of poly(acrylic acid) (PAAc) to RNase A. Complexation of RNase A with PEAMA-g-PEG induced the improvement of heat resistance of RNase A . Circular dichroism (CD) spectral analysis clearly indicated that the complexation of enzyme with polymer has almost no influence on the conformation of enzyme.

Associative and Dissociative Processes in Non‐Covalent Polymer‐Mediated Intracellular Protein Delivery

“…Questions surrounding cargo function and bioavailability post‐delivery are usually answered by delivering enzymes or other functional proteins and monitoring said function . In a separate body of non‐delivery‐related work, polymer–enzyme binding was studied with respect to turning enzyme function on and off . Taking advantage of this pre‐existing body of knowledge, one could image correlation studies in which polymer–protein binding constants were measured by using enzymes as cargo, for which the activity could be subsequently monitored both in and outside of the cell.…”

“…[35,54,57,94,95] In as eparate body of non-delivery-related work, polymer-enzymeb inding was studied with respect to turning enzyme function on and off. [66,92,96,97] Taking advantage of this pre-existing body of knowledge,o ne could image correlation studies in which polymer-protein binding constants were measured by using enzymesa sc argo,f or which the activity could be subsequently monitored both in and outside of the cell. Correlations could then be drawn between polymer bindingi nt he complex-formations tage and cargo activity in the release stage.…”