Improving the Heat Resistance of Ribonuclease A by the Addition of Poly(N,N‐diethylaminoethyl methacrylate)‐graft‐poly(ethylene glycol) (PEAMA‐g‐PEG)

Ganguli, Sumon; Yoshimoto, Keitaro; Tomita, Shunsuke; Sakuma, Hiroshi; Matsuoka, Tsuneyoshi; Shiraki, Kentaro; Nagasaki, Yukio

doi:10.1002/mabi.200900432

Cited by 6 publications

(12 citation statements)

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“…PEAMA-g-PEG is a novel modifying agent that prevents the inactivation and aggregation of certain types of heat-labile enzymes as proved in our previous published papers (Ganguli et al 2009;Ganguli et al 2010). It is hypothesized that the addition of only PEAMA-g-PEG improves the heat resistance of RNase A, due to the complex formation between RNase A and PEAMA-g-PEG by the combination of electrostatic and hydrophobic interaction (Ganguli et al 2010).…”

Section: Resultsmentioning

confidence: 92%

“…In addition, we evaluated the effect of using only PEAMA-g-PEG on the enzymatic activity and heat resistance of RNase A, which is composed of 124 amino acid residues, has a positive net charge at neutral pH (Ganguli et al 2010). According to X-ray crystallography results, the active site of RNase A has four positively charged residues, which play an important role in the enzymatic function of this enzyme (Borkakoti 1983).…”

Section: Resultsmentioning

confidence: 99%

“…These results are also in line with the previously reported data (Kurinomaru et al 2011). It should be noted that RNase A is composed of 124 residues and has a positive net charge of + 4 at pH 7.0 (pI= 11) (Ganguli et al 2010). Since it is well known that negatively charged polymers interact with positively charged enzymes effectively (Harada and Kataoka 2003), it is undoubtedly suggested that this negatively charged PAAc formed RNase A/PAAc complex substantially by electrostatic interaction.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, charged polymeric compounds are particularly attractive agents for complexation with enzymes. Recently, we succeeded in regulating the enzymatic activity and concomitantly improving the heat resistance of lysozyme (Ganguli et al 2010) through the external addition of a polycationic polyamine-poly (ethylene glycol) (PEG) graft copolymer, poly (N,Ndiethylaminoethyl methacrylate)-graft-PEG (PEAMA-g-PEG). Since switching the enzymatic activity is of great importance in various fields such as enzyme delivery and stabilization, in the present study, the simple and inexpensive procedure have been applied successfully in the case of another enzyme ribonuclease A (RNase A) with a anionic polymer poly (acrylic acid) (PAAc) and a cationic smart copolymer, poly (N,Ndiethylaminoethyl methacrylate)-graft-poly(ethylene glycol) (PEAMA-g-PEG).…”

Section: Introductionmentioning

confidence: 99%

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

Ganguli

2014

J bio-sci.

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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.

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Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Ganguli

2014

J bio-sci.

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“…L-Asparaginase is one of the therapeutic enzymes prescribed for acute lymphoblastic leukemia [91], with a tetrameric form of identical 35.6 kDa subunits [92,93]. An anionic stabilizes the cationic enzymes, lysozyme [96] and ribonuclease A [97] against heat treatment. This phenomenon is not so surprising because (i) PEG is a kind of aggregation suppressor, (ii) PEG has the salting-out effect, and (iii) a positively charged polyelectrolyte increases the propensity of repulsion between positively charged proteins.…”

Section: Stabilization Of Protein By Pegylated Polymermentioning

confidence: 99%

Wrap-and-Strip Technology of Protein–Polyelectrolyte Complex for Biomedical Application

Shiraki¹,

Kurinomaru²,

Tomita³

2016

CMC

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Different mechanisms of action of poly(ethylene glycol) and arginine on thermal inactivation of lysozyme and ribonuclease A

Tomita

Nagasaki

Shiraki

2012

Biotech & Bioengineering

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Proteins tend to undergo irreversible inactivation through several chemical modifications, which is a serious problem in various fields. We have recently found that arginine (Arg) suppresses heat-induced deamidation and β-elimination, resulting in the suppression of thermal inactivation of hen egg white lysozyme and bovine pancreas ribonuclease A. Here, we report that poly(ethylene glycol) (PEG) with molecular weight 1,000 acts as a thermoinactivation suppressor for both proteins, especially at higher protein concentrations, while Arg was not effective at higher protein concentrations. This difference suggests that PEG, but not Arg, effectively inhibited intermolecular disulfide exchange among thermally denatured proteins. Investigation of the effects of various polymers including PEG with different molecular weight, poly(vinylpyrolidone) (PVP), and poly(vinyl alcohol) on thermoinactivation of proteins, circular dichroism, solution viscosity, and the solubility of reduced and S-carboxy-methylated lysozyme indicated that amphiphilic PEG and PVP inhibit intermolecular collision of thermally denatured proteins by preferential interaction with thermally denatured proteins, resulting in the inhibition of intermolecular disulfide exchange. These findings regarding the different mechanisms of the effects of amphiphilic polymers--PEG and PVP--and Arg would expand the capabilities of methods to improve the chemical stability of proteins in solution.

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Improving the Heat Resistance of Ribonuclease A by the Addition of Poly(N,N‐diethylaminoethyl methacrylate)‐graft‐poly(ethylene glycol) (PEAMA‐g‐PEG)

Cited by 6 publications

References 39 publications

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

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

Wrap-and-Strip Technology of Protein–Polyelectrolyte Complex for Biomedical Application

Different mechanisms of action of poly(ethylene glycol) and arginine on thermal inactivation of lysozyme and ribonuclease A

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