Masahito Oka

Statistical analysis of the physical properties of the 20 naturally occurring amino acids

In order to describe the conformational and other physical properties of the 20 naturally occurring amino acid residues with a minimum number of parameters, several multivariate statistical analyses were applied to 188 of their physical properties and ten orthogonal properties (factors) were obtained for the 20 amino acids without losing the information contained in the original physical properties. The analysis consisted of three main steps. First, 72 of the physical properties were eliminated from further consideration because they did not pass statistical tests that they follow a normal distribution. Second, the remaining 116 physical properties of the amino acids were classified by a cluster analysis to eliminate duplications of highly correlated physical properties. This led to nine clusters, each of which was characterized by an average characteristic property, namely bulk, two hydrophobicity indices for free amino acids, one hydrophobicity index for amino acid residues in a protein, two types of β-structure preference, α-helix preference, and two types of bend-structure preference. The physical properties within a given cluster were highly correlated with each other, but the correlation between clusters was low. Third, a factor analysis was applied to the nine average classified properties and 16 additional physical properties to obtain a small number of orthogonal properties (ten factors). Four of these factors arise from the nine characteristic properties, and the remaining six factors were obtained from the 16 physical properties not included in the nine characteristic properties. Finally, most of the 188 physical properties could be expressed as a sum of these ten orthogonal factors, with appropriate weighting factors. Since these factors contain information relating almost all properties of all 20 amino acids, it is possible to estimate the numerical values of a property for one or two amino acids for which experimental data for this property are not available. For example, the estimated values for the Zimm-Bragg parameters at 20°C are 0.66 and 0.92 for proline and cysteine, respectively, computed from the first four factors.

Immobilization of bromelain onto porous copoly(γ-methyl-l-glutamate/l-leucine) beads

Abstract Water-insoluble bromelain was prepared by immobilizing bromelain onto the surface of porous copoly(γ-methyl- l -glutamate/ l -leucine) (ML) beads with and without spacer. The mode of the immobilization between bromelain and porous copolypeptide ML beads was covalent fixation. The relative activity and the stability of the immobilized bromelain was investigated. The retained activity of the bromelain covalently immobilized by the azide method was found to be excellent toward a small ester substrate, N -benzyl- l -arginine ethyl ester, but rather low toward casein, a high molecular weight substrate. The values of the Michaelis constant K m and the maximum reaction velocity V m for free and immobilized bromelain on the porous copolypeptide ML beads were estimated. Apparent K m was larger for immobilized bromelain than for the free one, while V m was smaller for the immobilized bromelain. The thermal stability of the covalently immobilized bromelain was higher than that of the free bromelain. The initial enzymatic activity of the immobilized bromelain remained approximately unchanged with storage time, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.

Enzymatic hydrolysis of random copolypeptides consisting of N-hydroxyethyl-L-glutamine and L-alanine, L-leucine, or L-valine

Abstract Two component water soluble random copolypeptides consisting of N-hydroxyethyl- l -glutamine and hydrophobic l -amino acid, such as l -alanine, l -leucine, or l -valine, were prepared by carrying out aminolysis reactions with 2-amino- l -ethanol (E) on starting copolymers consisting of γ-benzyl- l -glutamate (B) and the corresponding hydrophobic l -amino acid. The effects on copolymer composition and sequential distributions, as well as molecular conformations, on the rate of degradation by ficin in a pseudoextracellular fluid (PECF) at pH 7.4 and 37.0°C were investigated to simulate in vivo polymer degradation. All the samples were found to be extensively degraded by random chain fracture with ficin. Further, the degradation data for these samples followed the Michaelis–Menten rate law, being of the first order in ficin concentration. The nature of the side chains and the molecular conformations are important to the rate of degradation by ficin in vitro.

Theoretical Conformational Analysis on Silk Fibroin Model Polypeptide with Ala-Gly Repeated Sequence

Theoretical conformational analysis was carried out for Ac-(Ala-Gly)12-NHMe, which was a model polypeptide of Bombyx mori silk fibroin, using ECEPP and the conformational energy minimization procedure. The hypothesis on the interaction in polypeptide molecules was also used for the analysis. Calculated results showed that right-handed α-helix and left-handed β4.6-helix were the lowest-energy and 2nd low-energy conformations, respectively. Several stable conformations, which were related to the already proposed model structures of silk fibroin, were also found in the theoretically obtained conformational ensemble of Ac-(Ala-Gly)12-NHMe.

Spacer effects on enzymatic activity of bromelain immobilized onto porous chitosan beads

Abstract Bromelain was covalently immobilized onto the surface of porous chitosan beads with and without alkyl chain spacers of different lengths. The relative activity of the immobilized bromelain was found to be high toward a small ester substrate, N -benzyl- l -arginine ethyl ester (BAEE), but rather low toward casein, a high molecular weight substrate. Bromelain immobilized with spacer gave an almost constant activity by varying the surface concentration in marked contrast with the immobilized bromelain without spacer whose activity monotonously decreased with decreasing surface concentration. The relative activity of the immobilized bromelain for hydrolysis of a high molecular weight substrate was a strong function of spacer length. The pH, thermal, and storage stabilities of the immobilized bromelain were higher than those of the free bromelain. The bromelain immobilized directly onto the surface of chitosan beads without any spacer gave a higher stability than those immobilized with spacers. The spacer effect on the activity could be explained in terms of flexibility of the immobilized bromelain molecule.

Radiation sterilization of enzyme hybrids with biodegradable polymers

Abstract Ionizing radiations, which have already been utilized for the sterilization of medical supplies as well as gas fumigation, should be the final candidate to decontaminate “hybrid” biomaterials containing bio-active materials including enzymes because irradiation induces neither heat nor substances affecting the quality of the materials and our health. In order to check the feasibility of 60 Co-gamma rays on these materials, we selected commercial proteases including papain and bromelain hybridized with commercial activated chitosan beads and demonstrated that these enzyme-hybrids suspended in water showed the significant radiation durability of more than twice as much as free enzyme solution at 25-kGy irradiation. Enhanced thermal and storage stability of the enzyme hybrids were not affected by the same dose level of irradiation, either, indicating that commercial irradiation sterilization method is applicable to enzyme hybrids without modification.

Preparation and properties of biodegradable copoly(N-hydroxyalkyl-D, L-glutamine) membranes

Abstract Random copolypeptide (PHADLG) membranes consisting of N -hydroxyalkyl- d -glutamine (HADG) and N -hydroxyalkyl- l -glutamine (HALO) covering the whole range of d , l -copolymer composition were prepared by carrying out aminolysis reactions with 2-amino-1-ethanol (E) or 5-amino-1-pentanol (Pe), together with a crosslinking reaction with 1,8-octamethylenediamine (OMDA) on membranes of the starting random copolymers (PMDLG) consisting of γ -methyl- d -glutamate and γ -methyl- l -glutamate. The relationships between their bulk structure and membrane properties were investigated, such as the swelling ratio in water, tensile properties, water vapor permeability, and enzymatic degradation behavior of the membranes in a pseudo-extracellular fluid (PECF), as a function of d , l -copolymer composition. The tensile properties of d , l -copolymer membranes were highly dependent on the swelling ratio of PECF. Biodegradation of these membranes in vitro by bromelain indicated that degradation was a bulk rather than a surface phenomenon, and that the rate of degradation of membranes was also highly dependent on the hydrophobicity of side chains, d , l -copolymer composition as well as on the swelling ratios of the d , l -copolymer samples.

Preparation and properties of A–B–A tri-block copolymer membranes consisting of N-hydroxypropyl-l-glutamine as the A component and l-alanine as the B component

Abstract A–B–A type tri-block copolymer consisting of N -hydroxypropyl- l -glutamine as the A component and l -alanine as the B component as well as the corresponding random copolymers and homopolymers were prepared by carrying out an aminoalcoholysis reaction with 3-amino-1-propanol, together with a crosslinking reaction with 1,8-octamethylenediamine on membranes of the starting polymer membranes including γ-methyl- l -glutamate residue. It was shown that the effective crosslink density was proportional to the percent crosslinker in the reaction mixture. The relation between their bulk structure and membrane properties were investigated, such as the swelling ratio in water ( q ), tensile properties, and enzymatic degradation behavior of the membranes in a pseudo-extracellular fluid (PECF). The tensile properties of the hydrophilic membranes were highly dependent on q in PECF, and on the hydrophobic portions in molecular chains, whose behavior was typical of an elastomer. Biodegradation of the membranes in vitro by bromelain indicated that the degradation took place in bulk rather than on the surface, and that the rate of degradation was also highly dependent on q in the samples as well as on the hydrophobic portion of the membranes in PECF.

Biodegradation of random co-polypeptide hydrogels consisting of N-hydroxypropyl L-glutamine as one component

Abstract Two component random co-polypeptide hydrogels consisting of N -hydroxypropyl l -glutamine and l -alanine (Ala) or l -phenylalanine (Phe) were prepared by performing aminolysis reactions with 3-amino-1-propanol together with crosslinking reaction with 1,8-octamethylenediamine on hydrogels of the starting co-polymers consisted of γ-benzyl l -glutamate and Ala or Phe. The relationship between their bulk structure and properties was evaluated with regard to the swelling ratio in water ( q ), the rate of water vapor permeability ( V f ), tensile properties, and enzymatic degradation behaviors of hydrogels in a pseudo-extracellular fluid (PECF). The tensile property of the hydrogels was highly dependent on q in PECF, and on the hydrophobicity of the side chains. A relationship was obtained between the V f and q of hydrogels in PECF regardless of the differences in the nature of the side chains. Biodegradation of the hydrogels in vitro by bromelain indicated that degradation took place in bulk rather than on the surface, and that the rate of degradation was also highly dependent on q in the samples as well as on the hydrophobicity of the side chains of the samples.

Conformational analysis of periodic polypeptides

SummaryTheoretical conformational analysis was carried out for a periodic polypeptide composed of the repetitive Ala-Pro sequence, i.e., poly(Ala-Pro) using ECEPP and the conformational minimization procedure. Calculated results showed that a γ-helix is the most stable helical conformation of poly(Ala-Pro), and also that most of the stable helical conformations are other types γ-helices and β-helices with large value of the rise per residue. Obtained conformational preference of poly(Ala-Pro) indicates that the repetitive Ala-Pro sequence is a suitable amino-acid sequence for designing the rod-like backbone conformations of artificial proteins.