Affinity chromatography of trypsin and related enzymes. V. Basic studies of quantitative affinity chromatography.

A detailed study of the quantitative affinity chromatography of trypsin [EC 3.4.21.4] is reported here. Frontal chromatography using an enzyme solution of very low concentration on an affinity adsorbent gave the dissociation constant of the enzyme-immobilized ligand complex (Kd). Kd values determined under various conditions enabled us to discuss in detail the interaction of trypsin and affinity adsorbents (mainly Gly-Gly-Arg Sepharose). The pH dependence of Kd was consistent with that of the interaction of trypsin and product-type compounds. The effects of changes in temperature, ionic strength, dielectric constant, etc., were also studied. The Ki values of soluble competitive inhibitors can be determined by analysis of their effects on the elution volume of the enzyme. The values obtained were in good agreement with those obtained by kinetic analysis. The present method proved to be useful as a general procedure to investigate the interaction of a protein and a specific ligand.     

Affinity chromatography of trypsin and related enzymes. I. Preparation and characteristics of an affinity adsorbent containing tryptic peptides from protamine as ligands.

An absorbent for the affinity chromatography of trypsin [EC 3.4.21.4] (AP Sepharose) was prepared. The ligand was a mixture of oligopeptides (mainly di- and tripeptides) containing L-arginine as carboxyl termini, and was obtained from a tryptic digest of protamine. Trypsin was absorbed at relatively low pH (7-4), but was not absorbed at the optimum pH of catalysis (8.2). This was clearly explained on the basis of the pH dependence of the interaction of trypsin with its products. Inactivated trypsin, trypsinogen, and chymotrypsin were not absorbed. The absorption of active trypsin was interferred with by either benzamidine or urea. From these observations, it is evident that AP Sepharose is an affinity adsorbent. AP Sepharose was useful for purification of commercial bovine trypsin. A preliminary application for the purification of Streptomyces griseus trypsin was also successful.     

Quantitative studies of ion-exchange and affinity elution chromatography of enzymes

Quantitative studies on the binding of a variety of enzymes to CM-cellulose have been carried out, and the magnitude of the affinity elution effect in the presence of substrates of the enzymes has been determined. In most cases the weakening of binding in the presence of substrate corresponded closely to the amount expected as a result of the overall charge change, but in a few examples the effect was greater. Some calculations have been made demonstrating the range of strengths of interactions between enzyme and adsorbent, and the energy involved per charge on the protein molecule.     

Restricted diffusion of molecules in porous affinity chromatography adsorbents.

A restricted diffusion model is constructed and solved in order to study the permeability of large adsorbate molecules in the pores of affinity chromatography media, when the adsorbate molecules are adsorbed onto immobilized ligands. The combined effects of steric hindrance at the entrance to the pores and frictional resistance within the pores, as well as the effects of pore size distribution, pore connectivity of the adsorbent, molecular size of adsorbate and ligand, and the fractional saturation of adsorption sites (ligands), are considered. Affinity adsorbents with dilute and high ligand concentrations are examined, and the permeability of the adsorbate in porous networks of connectivity nT is studied by means of effective medium approximation (EMA) numerical solutions. As expected, the permeability of the adsorbate decreases as the size of the adsorbate and/or ligand molecule increases. The permeability also decreases when the fractional saturation of the ligands increases, as well as when the pore connectivity of the network decreases. The dependence of the permeability on the pore connectivity tends to be less marked in adsorbents with concentrated ligand than in porous media with dilute ligand concentration. The conditions are also presented for which the percolation threshold is attained in a number of different systems. The restricted diffusion model and results of this work may be of importance in studies involving the modeling, prediction of the dynamic behavior, design, and control of affinity chromatography (biospecific adsorption) systems employing porous adsorbents. The theoretical results may also have important implications in the selection of a ligand as well as in the selection and construction of an affinity porous matrix, so that the adsorbate of interest can be efficiently separated from a given solution. Furthermore, with appropriate modifications this restricted diffusion model may be used in studies involving the immobilization of ligands or enzymes in porous solids.     

Affinity chromatography: purification of bovine trypsin and thrombin

Bovine trypsin has been purified by affinity chromatography on agarose beads containing covalently bound p-aminophenylguanidine, p-aminobenzamidine, or m-aminobenzamidine. Bovine thrombin was purified on a m-aminobenzamidine-agarose column containing a high concentration of the inhibitor. The values of the inhibition constant, K_i, for these inhibitors were determined for both enzymes and found to be 5–10 times poorer for thrombin than for trypsin. Only those benzamidines with low K_i values and coupled in high concentration to the agarose matrix were satisfactory for thrombin purification. Affinity-purified trypsin and thrombin were both greater than 90% active as measured by active site titration.     

New affinity adsorbents containing deoxycytidine, deoxyadenosine, or deoxyguanosine and their interactions with deoxynucleoside-metabolizing enzymes

3'-(4-Aminophenyl phosphate) derivatives of deoxycytidine (dCyd), deoxyadenosine (dAdo), and deoxyguanosine ( dGuo ) were synthesized. The inhibitory effects of these compounds on mammalian and bacterial deoxynucleoside kinases and several other deoxynucleoside-metabolizing enzymes were examined. The same derivatives were coupled to carboxyl-terminal Sepharose CL-6B (3-8 mumol of ligand/mL of gel), and each of the resulting affinity adsorbents was tested with various partially purified enzymes. Reasonable correlation between the inhibitory effect of a soluble deoxynucleoside 3'-phosphate diester and affinity of the corresponding Sepharose adsorbent for the enzyme was observed. Among the three dCyd kinases examined, only the bovine mitochondrial enzyme was adsorbed onto the dCyd-Sepharose column and eluted biospecifically by 1 mM dCyd (1400-fold purification). Its Ki toward the dCyd derivative was relatively low (1.1 mM), whereas no measurable inhibition was seen with mammalian cytosol or bacterial enzymes that did not stick to the column. The Ki of the dAdo derivative toward three dAdo kinases was more than 5 mM in each case, and none of these were retained by dAdo-Sepharose. Among the other dAdo-metabolizing enzymes examined, nucleoside phosphotransferase from barley (Ki = 1.2 mM) was adsorbed to dAdo-Sepharose at pH 5.0 and was biospecifically eluted with dAdo or AMP after suppressing ionic binding by adjusting the pH to 6.0 (480-fold purification to homogeneity). Mammalian mitochondrial dGuo kinase (beef liver) showed the lowest Ki (0.16 mM) among the enzymes tested and was biospecifically purified with dGuo -Sepharose (2800-fold purification).(ABSTRACT TRUNCATED AT 250 WORDS)     

Design and study of peptide-ligand affinity chromatography adsorbents: application to the case of trypsin purification from bovine pancreas

The purification of trypsin from bovine pancreas was employed in a case study concerning the design and optimization of peptide-ligand adsorbents for affinity chromatography. Four purpose-designed tripeptide-ligands were chemically synthesized (>95% pure), exhibiting an Arg residue as their C-terminal (site P(1)) for trypsin bio-recognition, a Pro or Ala in site P(2), and a Thr or Val in site P(3). Each tripeptide-ligand was immobilized via its N-terminal amino group on Ultrogel A6R agarose gel, which was previously activated with low concentrations of cyanuric chloride (10.5 to 42.5 mumol/g gel). Well over 90% of the peptide used was immobilized. Three different concentrations were investigated for every immobilized tripeptide-ligand, 3.5, 7.0, and 14 mumol/g gel. The K(D) values of immobilized tripeptide-trypsin complexes were determined as well as the purifying performance and the trypsin-binding capacity of the affinity adsorbents. The K(D) values determined were in good agreement with the trypsin purification performance of the respective affinity adsorbents. The tripeptide sequence H-TPR-OH displayed the highest affinity for trypsin (K(D) 8.7 muM), whereas the sequence H-TAR-OH displayed the lowest (K(D) 38 muM). Dipeptide-ligands have failed to bind trypsin. When the ligand H-TPR-OH was immobilized via its N-terminal on agarose, at a concentration of 14 mumol/g gel, it produced the most effective affinity chromatography adsorbent. This adsorbent exhibited high trypsin-binding capacity (approximately 310,000 BAEE units/mL of adsorbent); furthermore, it purified trypsin from pancreatic crude extract to a specific activity of 15,200 BAEE units/mg (tenfold purification), and 82% yield. (c) 1997 John Wiley & Sons, Inc.     

Affinity chromatography of haemoproteins: 1. Synthesis of various imidazole containing matrices and their interaction with haemoglobin

The functional centre of haemoproteins is generally formed by an iron porphyrin and amino acid residues of he protein component. Some haemoproteins are able to bind imidazole to the iron of the prosthetic group. The synthesis of imidazole containing matrices is described and the affinity of haemoglobin as a model compound to these matrices has been studied. It was found that the lenght and structure of spacers as well as substituents at the imidazole ring are of critical importance: the adsorption of methaemoglobin shows two different kinds of protein matrix interaction: in case of a space length < 5 Å no interaction occurs for steric reasons; at a length > 5 Å adsorption takes place via complex formation between imidazole and iron of a prosthetic group independent of the linkage in 1- or 4(5)-position of the imidazole ring to the mtrix: the complex formation between imidazole and iron is the decisive step but is not solely responsible for the stability of the ocmplex: a hydroxyl group at the side chain near the imidazole decreases the adsorption drastically: large substituents at the imidazole ring disturb complex formation with the iron, but not the adsorption of haemoglobin; in the presence of a long spacer (> 20 A), hydrophobic interactions are predominantly responsible for the adsorption process and imidazole does not play any role.     

Effective elution of antibodies by arginine and arginine derivatives in affinity column chromatography

It has been shown that the recovery of monomeric antibodies from protein A affinity chromatography is enhanced significantly by using arginine as an eluent. To extend the applications of arginine to antibody purification and obtain an insight into the mechanism of arginine elution, we compared arginine with citrate, guanidine hydrochloride (GdnHCl), arginine derivatives, and other amino acids in protein A chromatography. We also applied arginine to elution of polyclonal antibodies (pAbs) in antigen affinity chromatography. As described previously, arginine was effective in eluting monoclonal antibodies IgG1 and IgG4. Two arginine derivatives, acetyl-arginine and agmatine, resulted in efficient elution at pH 4.0 or higher, and this was comparable to arginine. On the other hand, other amino acids, such as glycine, proline, lysine, and histidine, are much less effective than arginine under identical pH conditions. Whereas elution increased with arginine concentration, elution with citrate was insignificant in excess of 1 M at pH 4.3. Arginine was also effective in fractionation of pAbs using antigen-conjugated affinity columns. Although GdnHCl was also effective under similar conditions, the eluted material showed more aggregation than did the protein eluted by arginine.     

A study of hydrolysis of various synthetic peptides with gastrointestinal enzymes using thin-layer chromatography

Hydrolysis in vitro of alpha- and epsilon-peptide bonds of synthetic amino acids and peptide substrates,--models of protein fragments, with digestive enzymes was studied. The kinetics of hydrolysis was studied by quantitative thin-layer chromatography followed by densitometric analysis of the chromatographic patterns. The rate constants of hydrolysis of Phe-Lys, Gly-Lys dipeptides and their epsilon-acetyl and epsilon-succinyl derivatives with leucine aminopeptidase and pancreatic enzymes were calculated. epsilon-Acyl residues of the substrates failed to split off under these conditions. The digestive enzymes hydrolysed the alpha-peptide bonds adjacent to the acylated lysine. Hydrolysis of epsilon-acetyl substrates proceeded faster as compared to epsilon-succinyl derivatives.     

Syntheses of argininal semicarbazone containing peptides and their applications in the affinity chromatography of serine proteinases

Eight argininal semicarbazone containing peptides prepared by liquid phase synthesis were all found to be reversible inhibitors of model serine proteinases including trypsin and plasma kallikrein (PK). Among the peptides tested, those having a Lys residue at position P2 displayed the maximum binding potency towards PK. One of the peptides, Leu-enkephalin-argininal semicarbazone, a comparatively weak inhibitor, was chosen in order to develop an affinity-based purification protocol for PK. The affinity column was prepared by covalent attachment of the NH2-terminal moiety of the peptidyl semicarbazone to a solid-phase matrix bearing a spacer group. For efficient binding of PK, it was found necessary to optimize parameters like the concentration of inhibitor linked to the solid matrix, the ionic strength of the buffer used, the temperature and the pH. The majority of the bound enzyme could be recovered following elution with guanidine hydrochloride or benzamidine hydrochloride in a high salt buffer at pH 6.0. The usefulness of the affinity procedure towards the purification of other serine proteinases is also discussed.     

Derivatization of epoxy-activated agarose with various carbohydrates for the preparation of stable and high-capacity affinity adsorbents: Their use for affinity chromatography of carbohydrate-binding proteins

Two types of affinity adsorbents for lectins were prepared by new simple procedures. Both types of adsorbents had high ligand concentration and chemically stable linkage between ligand and Sepharose 4B. Oligosaccharide ligands were coupled by reductive amination with sodium cyanoborohydride to amino-Sepharose 4B prepared by amination of epoxy-activated Sepharose 4B. The glycamyl-Sepharose 4B thus obtained had particularly high adsorption capacities for lectins; lactamyl-Sepharose 4B, 58 mg/l ml of gel for peanut lectin; maltamyl-Sepharose 4B, 146 mg/ml for concanavalin A; and tetra-N-acetylchitotetraamyl-Sepharose 4B, 36 mg/ml for wheat germ agglutinin. Hexosamine was coupled by the aid of carbodiimide to carboxyl-Sepharose 4B prepared by succinylation of amino-Sepharose 4B. Galactosamine-Sepharose 4B adsorbed 145 mg soybean agglutinin/l ml gel. The columns turned from a semitransparent white to a milky white as they were saturated with lectins.     

"Affinity" chromatography of steroid-transforming enzymes with a non-steroidal ligand.

The chromatographic behaviour of an avian oestradiol-17 beta dehydrogenase, the 3(17) beta-hydroxy steroid dehydrogenase from Pseudomonas testosteroni and cortisone reductase from Streptomyces dehydrogenans was studied on columns of p-(phenoxypropoxy)aniline attached to CNBr-activated Sepharose. The ligand was effective in adsorbing the oestradiol dehydrogenase from a partially purified extract of chicken liver, and the cortisone reductase was perferentially retained when mixtures of the three dehydrogenases were applied to columns in 10mM-buffer. Under these conditions the 3(17)beta-hydroxy steroid dehydrogenase was eluted in the front, but was adsorbed in the presence of 3 M-KCl. beta-N-Acetylglucosaminidase present in the liver preparation was not retained by the ligand, whereas lactate dehydrogenase from rabbit muscle was adsorbed in a manner similar to the retention pattern found on affinity chromatography with 2',5'-ADP--Sepharose. The mean overall purification of the oestradiol dehydrogenase was 13-fold, with a mean recovery of 53%. p-(Phenoxypropoxy)aniline offers promise for the purification of steroid-transforming enzymes where elution with substrate or cofactor is not wanted. It is also suggested that the ligand may be of service in the purification of receptors of hormonal steroids.