Reversed-phase high-performance liquid chromatography of peptides of porcine pepsin prepared by the use of various forms of immobilized alpha-chymotrypsin.

Reversed-phase high-performance liquid chromatography (RP-HPLC) separation was used for the comparison of peptide maps of pepsin after its digestions by different forms of immobilized alpha-chymotrypsin. Porcine pepsin was hydrolysed with soluble alpha-chymotrypsin, with alpha-chymotrypsins glycosylated with lactose or galactose coupled to hydrazide derivative of cellulose, with alpha-chymotrypsin attached to poly(acrylamide-allyl glycoside) copolymer or to glycosylated hydroxyalkyl methacrylate copolymer Separon or to agarose gel Sepharose 4B. Efficiency of enzymatic protein cleavage with regard to peptide mapping of porcine pepsin has been examined by the use of alpha-chymotrypsins immobilized by different methods. Best results were achieved after hydrolysis with alpha-chymotrypsin immobilized on poly(acrylamide-allyl glycoside) copolymers. Alpha-chymotrypsin immobilized by this way has further three times higher relative specific activity in comparison with the soluble one. Modified alpha-chymotrypsin was not suitable for efficient pepsin cleavage.

Oriented immobilization of chymotrypsin by use of suitable antibodies coupled to a nonporous solid support.

In order to eliminate the kinetic limitation of chymotryptic hydrolysis of proteins due to diffusion, nonporous hydroxyalkyl methacrylate solid support was developed and used for oriented immobilization of chymotrypsin by means of suitable polyclonal antibodies. Nonporous microspheres were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in an alcohol-toluene mixture stabilized with cellulose acetate butyrate. The resulting particles were 1.2 microm in diameter and possessed narrow size distribution. After modification with adipic acid dihydrazide they contained 2 micromol of reactive groups available for coupling of anti-chymotrypsin antibodies. Prepared immunosorbent adsorbed 166.7 microg of chymotrypsin per 1 g of dry carrier. Immobilized chymotrypsin retained practically 100% of its native proteolytic activity. Kinetic parameters of catalysis by chymotrypsin immobilized via this way were improved due to the good steric accessibility of the enzyme active site for high-molecular-mass substrates, when digestion of proteins in batch experiments was used.

Hydrazide-functionalized poly(2-hydroxyethyl methacrylate) microspheres for immobilization of horseradish peroxidase.

Nonporous cross-linked poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (poly(HEMA-co-EDMA)) microspheres were prepared by dispersion polymerization of HEMA and EDMA. The polymerization was performed in toluene/2-methylpropan-1-ol in the presence of cellulose acetate butyrate as a steric stabilizer and dibenzoyl peroxide initiator. The particle size may be increased by decreasing the toluene/2-methylpropan-1-ol ratio and by increasing polymerization temperature. Adipohydrazide was attached to the microspheres activated with 2,4,6-trichloro-1,3,5-triazine. After periodate oxidation of its carbohydrate moieties, horseradish peroxidase was coupled to the hydrazide-functionalized poly(HEMA-co-EDMA) microparticles up to 7.3 microgram of enzyme/g of carrier without a significant loss of its activity. Immobilized peroxidase was found to be stable, retaining more than 97% of its initial activity when stored for 23 days after the preparation.

Oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function.

The advantages of oriented immobilization of biologically active proteins are good steric accessibilities of active binding sites and increased stability. This not only may help to increase the production of preparative procedures but is likely to promote current knowledge about how the living cells or tissues operate. Protein inactivation starts with the unfolding of the protein molecule by the contact of water with hydrophobic clusters located on the surface of protein molecules, which results in ice-like water structure. Reduction of the nonpolar surface area by the formation of a suitable biospecifc complex or by use of carbohydrate moieties thus may stabilize proteins. This review discusses oriented immobilization of antibodies by use of immobilized protein A or G. The section about oriented immobilization of proteins by use of their suitable antibodies covers immobilization of enzymes utilizing their adsorption on suitable immunosorbents prepared using monoclonal or polyclonal antibodies, preparation of bioaffinity adsorbent for the isolation of concanavalin A and immobilization of antibodies by use of antimouse immunoglobulin G, Fc-specific (i.e. specific towards the constant region of the molecule). In the further section immobilization of antibodies and enzymes through their carbohydrate moieties is described. Oriented immobilization of proteins can be also based on the use of boronate affinity gel or immobilized metal ion affinity chromatography technique. Biotin-avidin or streptavidin techniques are mostly used methods for oriented immobilization. Site-specific attachment of proteins to the surface of solid supports can be also achieved by enzyme, e.g., subtilisin, after introduction a single cysteine residue by site-directed mutagenesis.

Purification of anti-chymotrypsin antibodies for the preparation of a bioaffinity matrix with oriented chymotrypsin as immobilized ligand.

Polyclonal antibodies suitable for the oriented immobilization of chymotrypsin were prepared by chromatography on a bioaffinity matrix which had the enzyme immobilized through its active site to antilysin, covalently linked to bead cellulose. After periodate oxidation of their carbohydrate moieties, the isolated antibodies were coupled to a hydrazide derivative of bead cellulose. The periodate oxidation step, which led to greater efficiency and stability of the immunosorbent, had no deleterious effect on antibody activity as assessed by ELISA. Addition of chymotrypsin to the immunosorbent yielded an enzymically active bioaffinity matrix with the optimum molar enzyme/antibody ratio of 2.

Chymocell--a new wound cleansing agent.

A new wound cleansing agent consisting of bead cellulose with covalently, firmly bound proteolytic enzyme, chymotrypsin, was prepared. It was found that this preparation can be applied in the treatment of suppurating wounds of all types with very good results.

Detection of pathological changes of proteins by peptide mapping after protein digestion by use of oriented immobilized proteinases.

Diagnostic methods for detecting gastric diseases using chymotryptic digestion of pepsin are discussed. Peptide maps can be prepared using reversed-phase high-performance liquid chromatography. Batchwise chromatography by use of membranes with immobilized Tyr(I2) was used for the isolation of pepsin from gastric mucosa extract or from human blood serum. Enzymes immobilized using suitable antibodies or through their sugar moieties can be used for the preparation of peptide maps because such enzymes share good steric accessibility to their active binding sites and possess increased thermal stability. Biospecific adsorption of proteins to immunosorbents combines the simultaneous isolation of these enzymes with their oriented immobilization. Proteins were stabilized by hydrophilization through the attachment of saccharide residues containing galactose residues. These residues could be activated by oxidation using galactose oxidase and subsequently immobilized to hydrazide-containing solid supports.

Galactosylation as a tool for the stabilization and immobilization of proteins.

This paper presents a brief overview of the role that the carbohydrate moieties of biologically active glycoproteins play in the stabilization and oriented immobilization of these proteins on solid supports. The synthetic galactosylation of hydrophobic areas or their surroundings on the protein surface improves the structural stability of native proteins against inactivation by the interaction of water with hydrophobic clusters. The lowering of the degree solvation of tyrosine residues in galactosylated trypsin and the model substance N-carbobenzoxy-L-glutamyl-L-tyrosine was proved by Raman spectroscopy. D-Galactose residues can be selectively oxidized, either with periodate or enzymatically, and the aldehyde groups thus formed are used for the immobilization of glycoproteins on solid supports with hydrazide groups under mild conditions.

Carbohydrates as a tool for oriented immobilization of antigens and antibodies.

A biospecific sorbent for the isolation of ovalbumin antibodies was prepared by coupling of ovalbumin via its periodate-oxidized carbohydrate moiety to bead cellulose modified with adipic acid dihydrazide. The anti-ovalbumin IgG fraction isolated on this sorbent from immune rabbit serum contained only antibodies against protein determinants of ovalbumin. Thus, when these IgG were immobilized through their carbohydrate moieties to cellulose beads it became possible to prepare a biospecific sorbent for concanavalin A by oriented adsorption of ovalbumin. Ovalbumin was specifically adsorbed via its protein moiety and its carbohydrate part remained free for interaction with concanavalin A.

Influence of salts on the covalent immobilization of proteins to modified copolymers of 2-hydroxyethyl methacrylate with ethylene dimethacrylate.

In the study of the covalent immobilization of aminoacylase, thermitase, pepsin, trypsin, chymotrypsin, elastase, subtilisin, penicillinamidohydrolase, carboxypeptidase A, cystathionine-beta-synthase, and anticathepsin D-IgG to copolymers of 2-hydroxyethyl methacrylate and ethylene dimethacrylate (Separon HEMA) containing epoxy groups a marked influence of added salts on the immobilization efficiency was observed. Yields in covalently bound active enzymes were dependent on the concentrations and type of ions added, which can be arranged according to the Hofmeister series. At a distinct concentration, the salting-out ions cause a protein-matrix hydrophobic interaction which is a prerequisite for the covalent bond formation.

Methacrylate gels with epoxide groups as supports for immobilization of enzymes in pH range 3-12.

Glycidyl methacrylate gels are carriers suitable for attachment of enzymes and for use in affinity chromatography. Experiments on the coupling of glycyl-L-leucine and acetyl-L-leucine to these gels have shown a high pH-dependence of the bond formation between the support and the alpha-amino group (pH optimum 9.7); the coupling reaction between the epoxide group and the carboxyl group is practically pH-independent. Serum albumin and trypsin were attached to a greater extent in acidic than in alkaline media. The effects of time and temperature were also studied. The catalytic action of immobilized trypsin, as well as its use for affinity chromatography of trypsin inhibitor, were studied.

Affinity chromatography of proteases of hydroxyalkyl methacrylate gels with covalently attached inhibitors.

The efficient isolation of trypsin and chymotrypsin from a crude pancreatic extract was achieved by affinity chromatography on specific adsorbents prepared by coupling of both naturally occurring protease inhibitors and also synthetic low-molecular-weight protease inhibitors to hydroxyalkyl methacrylate gels. Specific sorbents prepared with synthetic inhibitors are stable and are suitable for the isolation of chymotrypsin and trypsin even on a large scale.

Reactive carriers of immobilized compounds.

Sphericanl macroporous reactive carriers capable of forming covalent bonds with amino acids and proteins were prepared by the suspension copolymerization of 2-hydroxyethyl methacrylate, ethylene dimethacrylate and p-nitrophenyl esters of methacrylic acid and methacryloyl derivatives of glycine, beta-alanine and epsilon-aminocaproic acid. The effect of the spacer length, pH and the type of the buffer used, concentration of reactive groups in the copolymer, concentration of the ligand and the participation of the hydrolytic and aminolytic reaction of p-nitrophenyl functional groups in the attachment of glycine, D,L-phenylalanine and serumalbumin was studied. Macroporous copolymers containing reactive functional groups can be used as active enzyme carriers, if their activity is not blocked by the presence of p-nitrophenol split off in the attachment reaction.

Affinity chromatography on hydroxyalkyl methacrylate gels. III. Adsorption of chymotrypsin to poly(hydroxyalkyl methacrylates) with covalently bound benzyloxycarbonyl-glycyl-D-phenylalanine and -D-leucine as function of pH and ionic strength.

Chymotrypsin is specifically adsorbed at low ionic strength and alkaline pH to hydroxyalkyl methacrylate gels with N-benzyloxycarbonylglycl-D-phenylalanine or N-benzyloxycarbonylglycyl-D-leucine attached through 1,6-hexanediamine. Chymotrypsin is not adsorbed either to the unmodified gel (Spheron) or to the gel with attached, 1,6-hexanediamine (NH2-Spheron). The adsorption of chymotrypsin to Z-Gly-D-Phe-NH2-Spheron was investigated as a function of pH and ionic strength. Trypsin is not adsorbed to this gel. Chymotrypsin isolated from a crude pancreatic extract by affinity chromatography on Z-Gly-D-Phe-NH2-Spheron had the same activity as the enzyme isolated on a column of Spheron, to which the naturally-occurring trypsin inhibitor had been coupled.

Isolation of aminopeptidase from Aspergillus flavus.

A mixture of aminopeptidase and neutral protease from the Aspergillus flavus mold obtained by chromatography on DEAE-Sephadex was fractionated by chromatography on the hydroxyalkyl methacrylate gel with chemically bonded 1,6 hexamethylene diamine and D-leucine. Aminopeptidase thus obtained was electrophoretically homogeneous. Conditions for chromatography were worked out allowing a one stage isolation of a highly active aminopeptidase sample directly from the alcoholic precipitate of the culture medium of the Aspergillus flavus mold.

SH-proteinase from bean Phaseolus vulgaris var. Perlicka.

An SH-proteinase (EC 3.4.22.-) has been isolated from beans of the species Phaseolus vulgaris var. Perlicka. The enzyme is homogeneous when subjected to disc electrophoresis, electrofocusing and sedimentation analysis. The molecular weight was determined as 26,000-28,000 by gel filtration, 30,850 +/- 1500 by sedimentation analysis and 26,930-27,410 by calculation from the amino acid composition (Lys20-21, His3, Arg9, Asp21-22, Thr13, Ser18, Pro12-13, Glu23-24, Gly30, Ala16, Cys/29, Val19, Met1, Ile10, Leu13, Tyr14, Phe6, Trp3). The N-terminal amino acid of the proteinase is isoleucine. The effect of concentration, time of hydrolysis, pH, temperature, cations, anions, urea and guanidine - HCl on the proteolytic activity of the SH-proteinase was studied.

Pepsin immobilized by covalent fixation to hydroxyalkyl methacrylate gels: preparation and characterization.

Insoluble active derivatives of pepsin (EC 3.4.23.1) were prepared by covalent binding of this enzyme to hydroxyalkyl methacrylate gels modified with 1,6-diaminohexane or epsilon-aminocaproic acid in an acid medium by means of water-soluble carbodiimide. The amount of attached enzyme, its proteolytic activity, pH activity curves of the preparations obtained and the time and pH dependence of their stability were determined.

Affinity chromatography on hydroxyalkyl methacrylate gels. II. Isolation of thiol-containing protein and peptide using mercurial derivatives of gels.

Absorption properties of hydroxyalkyl methacrylate gels containing various amounts of mercuri derivatives of p-acetaminophenoxyethyl methacrylates (Hg-APEMA) and methacrylanilide (Hg-MAA) were investigated by means of a reduced Ellman's agent (5,5'-dithiobis-2-nitrobenzoic acid) and papain. The optimum gel was used for the affinity chromatography of SH-protease from beans and for the isolation of a peptide with a free sulphhydryl group from the chymotryptic hydrolyzate of serum albumin.