Petr Štrop

Chromatography of biopolymers and their fragments on ion-exchange derivatives of the hydrophilic macroporous synthetic gel spheron.

New ion-exchange materials have been developed from the synthetic glycol methacrylate gel Spheron: carboxymethyl-Spheron (weakly acidic); phosphonyl-Spheron (medium acidic); sulphonyl-Spheron (strongly acidic); diethylaminoethyl-Spheron (Type 1, weakly basic; Type 2, medium to strongly basic). Information is presented on the chromatographic characteristics of these new, macroporous, hydrophilic and rigid ion-exchangers as determined by experiments on the separation of mixtures of proteins, peptides, amino acids, nucleic acids, oligonucleotides, and nucleotides. The possibility is discussed of the use of ion-exchangers of this type in high-performance liquid chromatography of biopolymers.

Hydrophobic interaction chromatography of proteins and peptides on spheron P-300

Abstract Hydrophobic interaction chromatography of proteins and peptides on Spheron P-300 has been studied. Proteins such as human serum albumin, chymotrypsinogen and lysozyme are retained by this support; the higher the concentration of the salt added, the more protein is retained. The sorption and desorption were examined quantitatively with lysozyme. The theoretical plate height and its dependence on temperature during hydrophobic interaction chromatography of the lysozyme were determined. The hydrophobic interaction of the support was demonstrated by hydrophobic interaction chromatography of the protein components of human serum proteins, hog pancreatic amylase extract, and a peptide mixture obtained from tryptic digests of lysozyme. The effect of pH on the elution of human serum albumin, chymotrypsinogen and lysozyme was observed. The fractionation of crude hog pancreatic amylase by gradient elution was used to demonstrate the effect of alcohols as polarity-reducing agents. The properties required of materials used for hydrophobic interaction chromatography of biopolymers are discussed.

Sub-site preferences of the aspartic proteinase from the human immunodeficiency virus, HIV-1.

A series of synthetic, chromogenic substrates for HIV‐1 proteinase with the general structure Ala‐Thr‐His‐Xaa‐Yaa‐Zaa∗Nph‐Val‐Arg‐Lys‐Ala was synthesised with a variety of residues introduced into the Xaa, Yaa and Zaa positions. Kinetics parameters for hydrolysis of each peptide by HIV‐1 proteinase at pH 4.7, 37°C and u = 1.0 M were measured spectrophotometrically and/or by reverse phase FPLC. A variety of residues was found to be acceptable in the P3, position whilst hydrophobic/aromatic residues were preferable in P1. The nature of the residue occupying the P2; position had a strong influence on k cat (with little effect on k m;β‐branched residues Val or Ile in this position resulted in considerably faster peptide hydrolysis than when e.g. the Leu‐containing analogue was present in P2.

Separation of α- and β-trypsin by hydrophobic interaction chromatography

Abstract The separation of α- and β-trypsin by means of hydrophobic chromatography on Spheron P 300 was investigated with respect to the separation conditions, i . e ., salt concentration, pH, temperature, sample loading, flow-rate and support particle size. The optimal conditions have been selected at low pH (3.0) where the autodigestion of trypsin is suppressed. This method based on different exposures of hydrophobic amino acid residues of α- and β-trypsin is rapid, simple and effective for both analytical- and preparative-scale separations.

Ion-exchange derivatives of spheron : I. characterization of polymeric supports

Abstract In order to characterize matrices suitable for the preparationof ion exchangers, three commercially available types of glycol methacrylate macroreticular gels, SpheronTM P-100, P-300 and P-1000 (particle size, 20–40 μm), have been characterized by their bulk rate, degree of swelling, working volume and inner surface area. Prior to modification, the gels were extracted with dilute acid, dilute alkali. 8 M urea, pyridine and hot organic solvents. Extracted and dried Spherons have been characterized by elemental analysis, and by the determination of the inner surface area, exclusion limit, specific pore volume, the most frequent pore diameter, specific unpenetrable volume, number of unpolymerized double bonds and capacity for small ions. The particle-size distribution of Spheron P-300 has also been determined. Porosimetric data obtained by nitrogen sorption and desorption measurements are compared with those obtined by mercury porosimetry. The inner structure of Spheron is discussed in relation to the experimentl results (electron microphotography and the course of thermal vacuum depolymerization, ion-exchange capacity after the highest attainable ionogenic substitution, unpenetrable volume and particle porosity). Extracted Spheron P-300 exhibits an advantageous, approximately level dependence of the height equivalent to a theoretical plate on the flow-rate within the range 25–280 ml/h. The suitability of a Spheron matrix for the preparation of ion exchangers is demonstrated.

Chromophoric and fluorophoric peptide substrates cleaved through the dipeptidyl carboxypeptidase activity of cathepsin B

The action of bovine spleen cathepsin B as a dipeptidyl carboxypeptidase on newly synthesized substrates of the type peptidyl-X-p-nitrophenylalanyl (Phe(NO2))-Y (X,Y = amino acid residue) or 5-dimethylaminonaphthalene-1-sulfonyl (Dns)-peptidyl-X-Phe(NO2)-Y was investigated. The kinetic parameters of hydrolysis of the X-Phe(NO2) bond were determined by difference spectrophotometry (delta epsilon 310 = 1600 M-1 cm-1) or by spectrofluorometry by following the five- to eightfold increase of Dns-group fluorescence with excitation at 350 nm and emission at 535 nm. The substrates were moderately sensitive to cathepsin B; kcat varied from 0.7 to 4 s-1 at pH 5 and 25 degrees C; Km varied from 6 to 240 microM. The very acidic optima of pH 4-5 are characteristic for dipeptidyl carboxypeptidase activity of cathepsin B. Bovine spleen cathepsins S and H had little and no activity, respectively, when assayed with Pro-Glu-Ala-Phe(NO2)-Gly. These peptides should be a valuable tool for routine assays and for mechanistic studies on cathepsin B.

Ion-exchange derivatives of spheron

Abstract The preparation of diethylaminoethyl derivatives of Spheron (DEAE-Spheron 300) having different capacities for small ions (nominal capacities 0.11, 0.26, 0.60, 1,23, 1.63, 2.05 and 2.20 mequiv./g) is described. Routes leading to a high degree of ionogenic substitution are discussed. The ion exchangers were characterized analytically and by titration curves, which indicate that the ion exchangers are homoionic. The chromatographic properties of the DEAE-Spherons were tested in the chromatography of serum albumin, of an artificial mixture of proteins (lysozyme, chymotrypsinogen and serum albumin), blood plasma, a mixture of peptides and of glutamic, aspartic and cysteic acids using an amino acid analyzer and also in the chromatography of adenosine phosphates. In all cases the best separations were obtained with anion exchangers having the highest nominal capacities. The effect of degree of ionogenic substitution on the decrease of the partial hydrophobicity of the Spheron matrix is discussed. It is shown that at a nominal capacity exceeding 1.2 mequiv./g the effect of hydrophilic iogenic groups balances the partial hydrophobicity of the Spheron matrix, so that no hydrophobic sorption of proteins takes place.

Reduced-bond tight-binding inhibitors of HIV-1 protease Fine tuning of the enzyme subsite specificity

Truncation of a peptide substrate in the N‐terminus and replacement of its scissile amide bond with a non‐cleavable reduced bond results in a potent inhibitor of HIV‐1 protease. A series of such inhibitors has been synthesized, and S2–S3′ subsites of the protease binding cleft mapped. The S2 pocket requires bulky Boc or PIV groups, large aromatic Phe residues are preferred in P1 and P′ and Glu in P2′. The S3′ pocket prefers Phe over small Ala or Val. Introduction of a Glu residue into the P2′ position yields a tight‐binding inhibitor or HIV‐1 protease, Boc‐Phe‐[CH2‐NH]‐Phe‐Glu‐Phe‐OMe, with a subnanomolar inhibition constant. The relevant peptide derived from the same amino acid sequence binds to the protease with a K i of 110 nM, thus still demonstrating a good fit of the amino acid residues into the protease binding pockets and also the importance of the flexibility of P1‐P1′ linkage for proper binding. A new type of peptide bond mimetic, N‐hydroxylamine ‐CH2‐N(OH)‐, has been synthesized. Binding of hydroxylamino inhibitor of HIV‐1 protease is further improved with respect to reduced‐bond inhibitor.

Rapid chromatographic separation of technical enzymes on spheron ion exchanges

Abstract New types of ion exchangers with a hydroxyalkyl methacrylate matrix (Spheron) that are suitable for the the sorption and high-performance liquid chromatography of technical enzymes are described. Their use is illustrated by examples of the sorption and desorption of a microbial protease and by examples of the rapid, semi-preparative chromatography of technical enzymes (protease, glucose oxidase, pectinase).

Model study of hydrophobic interactions of α- and β-trypsin and α-chymotrypsin

Abstract The hydrophobic interactions of α- and β-trypsin as a function of ionic strength and pH were studied by hydrophobic chromatography. Evidence was obtained that in spite of the identical specificities and similar activities of α- and β-trypsin, the cleavage of the Lys—Ser bond induces conformational changes in the neighbourhood of the active site. Over a wide range of pH and salt concentration the non-polar residues on the surface of the molecule of β-trypsin are more exposed to an external environment than on the molecule of α-trypsin. In the trypsin(chymotrypsin)—inhibitor complexes the majority of hydrophobic amino acids are buried; other hydrophobic residues localized on the surface contribute only very slightly to the interaction with the chromatographic support. The retention of trypsin, chymotrypsin and their diisopropylphosphoryl derivatives on a support with flexible hydrophobic ligands bonded to the matrix through a spacer (octyl-Sepharose) was correlated with the retention on a support with hydrophobic binding sites incorporated into the rigid matrix of the resin (Spheron). The native enzymes are always more retained; this indicates that the substitution results in the shielding of the non-polar residues in the neighbourhood of the active site. The differences in the slope of individual proteins, resulting from the correlation of the retention values obtained with both supports at several sodium chloride concentrations are explained by differences in the accessibility of the surface non-polar residues in the individual proteins. In experiments with model peptides the contribution of the individual hydrophobic amino acids to the retention was investigated.