Yoh Kodera

Biomedical and biotechnological applications of PEG- and PM-modified proteins

Chemical modification of proteins and other bioactive molecules with polyethylene glycol (PEG) or its derivatives (PM) can be used to tailor molecular properties to particular applications, eliminating disadvantageous properties or conferring new molecular functions. Complexes of therapeutic proteins and PEG or PM show reduced immunoreactivity, prolonged clearance times and improved biostability. Modification with PEG can also increase the solubility and activity of enzymes in organic solvents, thus extending their potential for application in organic syntheses and biotransformation processes.

Cell cycle arrest and apoptosis of leukemia cells induced by L -asparaginase

Apoptotic cell death of murine leukemia cells induced by E. coli L-asparaginase was studied. Deprivation of L-asparagine from the culture of L5178Y cells by L-asparaginase caused the fragmentation of chromosomal DNA of the leukemia cells within 24 h. Prior to the degradation of DNA, cell cycles of L5178Y cells were found to be arrested in G1 phase, and evidence of the DNA strand breaks was initially observed in G1 phase cells as early as 8 h after the asparaginase treatment. Therefore, apoptosis of leukemia cells induced by L-asparaginase is an event that is associated with the cell cycle arrest in G1 phase.

Bioconjugates of proteins and polyethylene glycol : potent tools in biotechnological processes

Abstract Chemical modification of enzymes and other bioactive molecules with polyethylene glycol derivatives, activated PEG and PM, can eliminate some of the drawbacks of the biomolecules and/or give them new functions in biotechnological processes. PEG- or PM-lipase becomes soluble and active in organic solvents so that the reverse reactions of hydrolysis proceed effectively, not only in organic media but also in straight substrates without any solvent. These include ester synthesis and ester exchange reactions including lactone synthesis and optical resolution Enzymes such as lipase and asparaginase modified with activated PMs gain stabilization towards heat and urea denaturation and, for asparaginase in vivo, prolongation of clearance time. Photostabilization of natural pigments, magnetization of enzymes and effective affinity partitioning are archieved by modification with PEG derivatives.

Activation of Kallikrein-Kinin System in Human Plasma with Purified Serine Protease from Dermatophagoides farinae

An aqueous extract from a mite culture, of Dermatophagoides farinae, activated prekallikrein to kallikrein in normal plasma. Crude protein preparation, obtained by ammonium sulfate precipitation (95% saturation) from the extract, exhibited high activity (0.81 units/mg protein) towards a synthetic substrate of coagulation factor XIIa, Boc-Gln-Gly-Arg-MCA, and had also activity to form kallikrein in human plasma deficient in coagulation factor XII. Treatment of the protein preparation with phenylmethylsulfonyl fluoride (PMSF), an inhibitor of serine enzyme, gave rise to inactivation of both activities. Thus, the serine protease specific for Boc-Gln-Gly-Arg-MCA in mite cultures of D. farinae was purified by ammonium sulfate precipitation and chromatographies on p-aminobenzamidine-sepharose CL-4B, DEAE-Toyopearl 650M, Sephadex G-75 superfine and Sephacryl S-200. The purified protease was homogeneous electrophoretically, and its molecular weight was estimated to be 30,000. The optimum pH and temperature were around 7.5 and 50 degrees C, respectively. The specific activity was 36 units/mg protein at pH 7.4 and 37 degrees C. The activity was completely inhibited by PMSF. The serine protease had the activity to activate the kallikrein-kinin system in normal human plasma.

Review : Polyethylene Glycol(PEG)-Protein Conjugates: Application to Biomedical and Biotechnological Processes:

Proteins can be modified chemically by attaching synthetic or natural macromolecules to the surface of the protein molecule. Polyethylene glycol has been applied to various enzymes as a superior agent for modification. Organic solvent-soluble enzyme can be prepared by covalently attaching enzymes with activated PEG. PEG-enzyme conjugates have been coupled to magnetite, Fe 3 O 4 to form magnetic enzymes. These types of approaches may have a practical potential in the biomedical and biotechnological area.

Anti-inflammatory activity of superoxide dismutase conjugated with sodium hyaluronate

Superoxide dismutase (SOD) from bovine erythrocytes was conjugated with sodium hyaluronate (HA) with a mean molecular weight of 106 to have greater anti-inflammatory activity in vivo. Amino groups of SOD were coupled with carboxyl groups in the hyaluronate molecule using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The HA-SOD conjugate was composed of 1.5 mol of SOD molecule per 1 mol of hyaluronate on the average, and retained 70% of the activity of unmodified SOD. The conjugate was essentially non-immunogenic in mice, and exhibited much higher anti-inflammatory activities than HA or SOD in models of inflammatory diseases such as ischemic oedema of the foot-pad in mice, carrageenin-induced pleurisy and adjuvant arthritis in rats.

Chemical modification of L-asparaginase with a comb-shaped copolymer of polyethylene glycol derivative and maleic anhydride

L-Asparaginase from Escherichia coli, an anti-tumor enzyme, was chemically modified with two types of maleic anhydride copolymers with a comb-shaped form, the one composed of polyoxyethylene allyl methyl diether with the molecular weight of 13,000 (activated PM13) and the other of polyoxyethylene 2-methyl-2-propenyl methyl diether with 100,000 (activated PM100). The modified asparaginases (PM13- and PM100-asparaginases) exhibited the complete loss of immunoreactivity towards anti-asparaginase serum. The enzymic activity of PM100-asparaginase without immunoreactivity was well retained by 85% of non-modified one, while that of PM13-asparaginase was retained 46%. These results were discussed in relation to the chemical structure of modifying reagents including chain shaped-polyethylene glycol derivatives.

[7] Modification of proteins with polyethylene glycol derivatives

Publisher Summary This chapter discusses the chemical modification of proteins with synthetic macromolecules— polyethylene glycol (PEG) derivatives. The purposes of these modifications include alteration of immunoreactivity, immunogenicity, and suppression of immunoglobulin E production, or making enzymes soluble and active in organic solvents. Proteins can be modified with an activated PEG derivative. The methods of activation of polyethylene glycol were illustrated by Harris. The modifier is usually synthesized from monomethoxyPEG that has a hydroxy group at one end of the molecule amenable to manipulation. The chapter describes the syntheses of the modifiers. Most of the modifiers have a chain-shaped form, such as 2,4-bis( O -methoxyPEG)-6-chloro-s-triazine, abbreviated as “activated PEG 2 .” The chapter describes a new type of modifier with a comb-shaped form, which is a copolymer of maleic anhydride and a monomethoxyPEG derivative, abbreviated as “activated PM.” Each modifier reacts mainly with the ɛ-amino group of lysine residues and/or the N-terminal amino group.

Isolation of Biopterin-α-glucoside from Spirulina (Arthrospira) platensis and Its Physiologic Function

Abstract: A fluorescent substance was isolated from the cyanobacterium with a yield of 4.5 mg per 10 g of dried Spirulina (Arthrospira) platensis cells by gentle extraction and ethanol fractionation followed by column chromatography. The fluorescent substance, which has absorption maxima at 256 nm and 362 nm (pH 8.4), was identified as biopterin-α-glucoside by spectrophotometry and nuclear magnetic resonance spectroscopy. Biopterin-α-glucoside prevented decolorization of the photosynthetic pigments, chlorophyll a, phycocyanin, and carotenoids in photosynthetic vesicles of Spirulina platensis cells, by ultraviolet irradiation.

Chemical modification of lipase with a comb-shaped synthetic copolymer of polyoxyethylene allyl methyl diether and maleic anhydride

SummaryLipase fromPseudomonas fluorescens was coupled with a copolymer of polyoxyethylene allyl methyl diether and maleic anhydride, activated PM. The PM-lipase became soluble and active in organic solvents, and also heat stable. It catalyzed the ester synthesis in benzene and ester hydrolysis in an aqueous system with high enzymic activity.