Affinity chromatographic sorting of carboxypeptidase a and its chemically modified derivatives

Carboxypeptidase A and derivatives obtained by chemical modification of various active center components were subjected to affinity chromatography on a p-aminobenzylsuccinic acid-Sepharose 4B conjugate. Tetardation of the enzyme on the column was dependent on the residue modified when elution was carried out with 0.3 m NaCl at pH 7.0. Both the functional zinc atom and the active site residue Glu-270 are essential for effective adsorption while alteration of residues involved in hydrophobic interaction with substrate or in recognition of its terminal carboxyl group decreased retention on the affinity matrix. Elution of native carboxypeptidase with competing soluble benzylsuccinic acid indicated that only active center binding of the immobilized inhibitor accounts for retardation of the enzyme on the column. Hence, affinity chromatography on this biospecific adsorbent using mild elution conditions (which do not distort protein structure) provides an excellent tool for the rapid isolation and purification of active center modified enzyme even from a complex mixture of reaction products.     

Anhydrotrypsin: new features in ligand interactions revealed by affinity chromatography and thionine replacement.

Anhydrotrypsin was isolated in high purity from the product of base elimination from phenylmethanesulfonyl-trypsin, by a single operation of affinity chromatography. The adsorbent used for the chromatography was an agarose derivative coupled with peptides containing C-terminal arginine residues. As the affinity of the adsorbent for anhydrotrypsin was high compared with that for trypsin, purification of the enzyme derivative was easily achieved without the prior inactivation of trypsin which had been regenerated during the elimination reaction. Comparative studies of the ligand interaction specificities with anhydrotrypsin and trypsin confirmed the stronger interaction of the former protein with product-type ligands such as Bz-Arg-OH. No marked differences were observed between them in affinities toward substrate-type ligands such as Bz-Arg-NH2. The higher affinity of anhydrotrypsin was found to be limited to product-type ligands of L-configuration, i.e., the protein displayed an ability to discriminate the L-ligand from its optical isomer. THE PKa value for the ionization form of anhydrotrypsin responsible for the interaction with Bz-Arg-OH was estimated to be 7.60+/-0907     

Sepharose-bound trypsin and activated sepharose-bound trypsinogen. Studies with small and large substrates

Several enzymic and physical properties of Sepharose-bound trypsin and activated Sepharose-bound trypsinogen have been compared to those of the soluble enzyme. Sepharose-bound trypsinogen could be activated to the same extent as soluble trypsinogen; the release of the activation peptide and formation of the active site occurred as expected in the presence of catalytic amounts of trypsin. With synthetic substrates, the relative activity and pH dependence of both immobilized trypsin preparations were essentially identical and nearly the same as the soluble enzyme. Sepharose-trypsin also formed an inactive complex with soybean trypsin inhibitor, with 85% of the active sites participating. In contrast, the activity of Sepharose-trypsin with chymotrypsinogen and with trypsinogen as substrates was only 40% that of soluble trypsin. There is evidence for some catalytic heterogeneity of active sites of bound trypsin; probably those sites buried within the gel have a limited catalytic efficiency with macromolecular substrates. The immobilized enzyme is more stable than the soluble enzyme at elevated temperatures and to concentrated urea, and denaturation by urea at pH 8 is fully reversible since the loss of molecules by autolysis is eliminated.     

Glucosidase I, a transmembrane endoplasmic reticular glycoprotein with a luminal catalytic domain

We have analyzed the functional domain structure of rat mammary glucosidase I, an enzyme involved in N-linked glycoprotein processing, using biochemical and immunological approaches. The enzyme contains a high mannose type sugar chain that can be cleaved by endo-beta-N-acetyl-D-glucosaminidase H without significantly affecting the catalytic activity. Based on trypsin digestion pattern and the data on membrane topography, glucosidase I constitutes a single polypeptide chain of 85 kDa with two contiguous domains: a membrane-bound domain that anchors the protein to the endoplasmic reticulum and a luminal domain. A catalytically active 39-kDa domain could be released from membranes by limited proteolysis of saponin-permeabilized membranes with trypsin. This domain appeared to contain the active site of the enzyme and had the ability to bind to glucosidase I-specific affinity gel. Phase partitioning with Triton X-114 indicated the amphiphilic nature of the native enzyme, consistent with its location as an integral membrane protein, whereas the 39-kDa fragment partitioned in the aqueous phase, a characteristic of soluble polypeptide. These results indicate that glucosidase I is a transmembrane protein with a luminally oriented catalytic domain. Such an orientation of the catalytic domain may facilitate the sequential processing of asparagine-linked oligosaccharide, soon after its transfer en bloc by the oligosaccharyl transferase complex in the lumen of endoplasmic reticulum.     

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.     

Cell surface galactosyltransferase: immunochemical localization

A cell surface UDP-galactose:N-acetylglucosamine galactosyltransferase (GT) has been directly localized on bovine cells in tissue culture by immunohistochemical techniques. A conventional rabbit heteroantiserum was prepared against an affinity-purified soluble form of GT from bovine milk, and a monospecific IgG fraction was isolated by affinity chromatography on a GT adsorbent. As demonstrated by indirect immunofluorescence, antigen to this antibody is present on the surface of all three bovine cell lines tested. It was uniformly distributed over the exposed membrane surface of fixed cells. Exposure of living cells to the anti-GT antibody resulted in its time-dependent aggregation in the plane of the membrane. Antigen (GT) was released from the membrane surface by trypsin digestion, and its reappearance required protein synthesis, since cycloheximide effectively prevented repopulation of the cell surface.     

Microscale affinity purification of trypsin reduces background peptides in matrix-assisted laser desorption/ionization mass spectrometry of protein digests

The use of trypsin for protein digestion is hampered by its autolysis and low thermostability. Chemical modifications have been employed to stabilize the enzyme. Modified trypsin (e.g. methylated) usually enables performing digestions at elevated temperatures, but it still produces autolytic peptides. In this work, unmodified bovine trypsin was subjected to a microscale affinity chromatography on Arginine Sepharose (ASE) or Benzamidine Sepharose (BSE), which utilized the principle of active-site ligand binding. Trypsin was retained on the sorbents in ammonium bicarbonate as a binding buffer. After washings to remove unbound impurities, the enzyme was eluted by arginine as a free ligand (from ASE) or by diluted hydrochloric acid (from BSE). MALDI-TOF mass spectrometry confirmed removal of large molecular fragments as well as autolytic and other background peptides. Consequently, the purified trypsin was tested for its performance in procedures of in-gel digestion of protein standards and selected urinary proteins from real samples. It has been shown that the affinity purification of trypsin decreases significantly the number of unmatched peptides in peptide mass fingerprints. The presence of arginine in the digestion buffer was found to reduce intensity of autolytic peptides. As a result, the described purification procedure is applicable in a common proteomic routine.     

Structural features of CD4 required for binding to HIV

A soluble form of the human CD4 glycoprotein (sCD4), the cellular receptor for human HIV, was treated with various physical, chemical, and enzymic regimens and tested over a range of concentrations for its capacity to inhibit the binding of HIV to CD4+ T cells. Reduction of disulfide bonds and alkylation in denaturing buffer (8 M urea) destroyed the inhibitory activity of sCD4, whereas reduction and alkylation in PBS had no effect. Derivatization or digestion of carbohydrate groups by periodate oxidation or by glycolytic enzyme digestion did not affect sCD4 inhibitory capacity. Digestion with trypsin or endoproteinase Glu-C destroyed activity. A limited digestion of sCD4 with endoproteinase Glu-C resulted in a mixture of fragments, however, and the mixture had inhibitory activity equivalent to that of intact sCD4. Within this mixture, a fragment of 23 kDa was identified that binds to HIV. Although sCD4 can be digested to yield fully active fragments, the requirement for intrachain disulfide bonding indicates that the minimum sized portion of CD4 that will retain full affinity for HIV will have to be formulated with a proper tertiary structure.     

Isolation and characterization of a trypsin fraction from the pyloric ceca of chinook salmon (Oncorhynchus tshawytscha)

A trypsin fraction was isolated from the pyloric ceca of New Zealand farmed chinook salmon (Oncorhynchus tshawytscha) by ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The chinook salmon enzyme hydrolyzed the trypsin-specific synthetic substrate benzoyl-dl-arginine-p-nitroanilide (dl-BAPNA), and was inhibited by the general serine protease inhibitor phenyl methyl sulfonyl fluoride (PMSF), and also by the specific trypsin inhibitors — soybean trypsin inhibitor (SBTI) and benzamidine. The enzyme was active over a broad pH range (from 7.5 to at least pH 10.0) at 25 °C and was stable from pH 4.0 to pH 10.0 when incubated at 20 °C, with a maximum at pH 8.0. The optimum temperature for the hydrolysis of dl-BAPNA by the chinook salmon enzyme was 60 °C, however, the enzyme was unstable at temperatures above 40 °C. The molecular mass of the chinook salmon trypsin was estimated as 28 kDa by SDS–PAGE.     

Active site amino acid sequence of the bovine O6-methylguanine-DNA methyltransferase.

An O6-methylguanine-DNA methyltransferase has been partially purified from calf thymus by conventional biochemical techniques. The enzyme was specifically radioactively labelled at the cysteine residue of the active site and further purified by attachment to a solid support. Following digestion with trypsin, a radioactive peptide containing the active site region of the protein was purified by size fractionation, ion exchange chromatography and reverse phase HPLC. The technique yielded an essentially homogeneous oligopeptide which was subjected to amino acid sequencing. The sequence adjacent to the acceptor cysteine residue of the bovine protein exhibits striking homology to the C-terminal methyl acceptor site of the E. coli Ada protein and the proposed acceptor sites of the E. coli Ogt and the B. subtilis Dat1 proteins.     

The continuous isolation of trypsin by affinity adsorbent recycling

A method for the continuous affinity separation of proteins is described in which the adsorbent, in the form of a polymer belt, is recycled through feedstock and eluent liquid flows. As the belt is nonporous, contact between the solute and the ligand is not diffusion-dependent. Consequently, rapid cycle rates are possible. Soybean trypsin inhibitor immobilized on nylon was used as an affinity ligand for the isolation of trypsin. During a 30-h continuous run, trypsin was isolated from a crude preparation of bovine pancreas with a recovery of 30% to 40%. Approximately 18 mg of trypsin was obtained from 500 mg of protein using a total of approximately 10 mug of ligand. Electrophoretic analysis of the eluent showed that chymotrypsin, which also binds to SBTI, was the only major contaminant of the product. It was demonstrated that the highest rates of protein purification were obtained using solid/liquid contact times well below that required to achieve saturation of the affinity adsorbent. Slower adsorbent recycle rates, which achieved higher protein binding per unit area of belt, resulted in lower protein purification per unit time. The rate of purification was also dependent on the concentration of target protein in the adsorption chamber at steady state. As high concentrations increased losses from the chamber outflow, this resulted in a compromise between throughput and recovery during the adsorption phase. Under the conditions investigated, recoveries of over 60% were obtained, and a maximum throughput of approximately 2.5 mg trypsin per hour was achieved. Preliminary studies have shown that this can be improved by compartmentalizing the adsorption chamber, which can reduce losses from the adsorption chamber to less than 5%. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 538-545, 1997.     

Silanized silica bound trypsin as analytical probe.

Trypsin immobilized by covalent coupling to silanized silica shows significant activity (30-38%) and greater thermostability as compared to soluble trypsin. Proteolytic processing of albumin at varying periods suggest that the enzyme matrix can be used efficiently for limited proteolysis. Repeated use of the immobilized enzyme in protein digestion produces similar products as seen by electrophoretic analysis. Also, digestion of albumin by the immobilized enzyme follows similar pattern as that by soluble enzyme. The enzyme matrix can be easily removed from the incubation mixture. The results indicate the possibility of the immobilized enzyme for its effective application as analytical tool in peptide mapping and limited proteolytic processing.     

An efficient trypsin digestion strategy for improving desB30 productivity from recombinant human insulin precursor fusion protein

The insulin precursor (IP) expressed in Pichia pastoris is a single-chain peptide fused with a spacer peptide (EEAEAEAEPK) localized at its N-terminus and containing three trypsin cleavage sites in the polypeptide chain. The IP fusion protein is trypsinized to generate the insulin product desB30, which has a deletion of threonine^B30. The three restriction sites on IP fusion protein had different affinities for trypsin and were digested in sequential order. Further analysis showed that approximately 20% of the IP digestion intermediates could not be converted into the final desB30 product if the IP fusion protein was digested in an aqueous phase. This result can be attributed to the formation of IP dimers or hexamers, which could restrict enzyme reactivity in the aqueous phase. To enhance the conversion yield of the IP fusion protein to desB30 products, a new digestion method was established. The IP was digested in the eluent that resulted from reverse phase chromatography during the purification process, which improved the yield of digestion from 80.2% to 95.6%.     

C-terminal processing of tyrosinase is responsible for activation of Pholiota microspora proenzyme

Tyrosinase is expressed as a 67-kDa protein in Pholiota microspora (synonym Pholiota nameko), whereas the same enzyme purified from fruiting bodies of P. microspora is a 42-kDa protein that is cleaved with a C-terminal 25-kDa polypeptide from the 67-kDa protein. To confirm the role of C-terminal processing in enzyme activity, we expressed a recombinant 67-kDa tyrosinase in Escherichia coli cells. To obtain a soluble protein, the recombinant tyrosinase was expressed as a thioredoxin fusion protein with an enterokinase-cleavable site. Enterokinase digestion of the fusion protein produced a recombinant 67-kDa tyrosinase that did not have any catalytic activity. However, chymotrypsin digestion of the fusion protein produced a recombinant 44-kDa tyrosinase that was catalytically active and had a 25-kDa cleaved C-terminal. Kinetic parameters of the 44-kDa tyrosinase were similar to those of the 42-kDa tyrosinase purified from the fruiting bodies. These results suggest that tyrosinase is expressed in P. microspora as a latent 67-kDa proenzyme and is converted to the mature active 42-kDa enzyme by proteolytic processing of the C-terminal.     

Optimisation and validation of an angiotensin-converting enzyme inhibition assay for the screening of bioactive peptides

Angiotensin-converting enzyme (ACE) plays a major role in the regulation of blood pressure. A diagnostic assay to measure angiotensin-converting enzyme (ACE) activity was transformed into an enzyme inhibition assay and optimised, which led to a more sensitive and less expensive assay. By this spectrophotometric method, ACE inhibition is measured using the substrate furanacryloyl-Phe-Gly-Gly and as ACE source rabbit lung acetone extract. The optimised as well as the original ACE inhibition assay were used to verify the ACE inhibitory activity of captopril. The ACE inhibition assay was further validated by enalapril, its active derivative enalaprilat and the ACE-inhibitory peptide Ala-Leu-Pro-Met-His-Ile-Arg, corresponding to a tryptic fragment of bovine beta-lactoglobulin. Sigmoid curves could be fit adequately to the data points representing ACE inhibition in function of inhibitor concentration. IC(50) values for these compounds corresponded well with literature data. Furthermore, pea and whey protein hydrolysates obtained by digestion with trypsin showed ACE inhibitory activity in the ACE inhibition assay. Hence, this optimised assay is suitable to screen for ACE inhibitory peptides derived from food proteins with a possible antihypertensive effect in vivo.     

Application of an acid proteinase from <Emphasis Type="Italic">Monascus purpureus</Emphasis> to reduce antigenicity of bovine milk whey protein

An acid proteinase from Monascus purpureus No. 3403, MpuAP, was previously purified and some characterized in our laboratory (Agric Biol Chem 48:1637–1639, 1984). However, further information about this enzyme is lacking. In this study, we investigated MpuAP’s comprehensive substrate specificity, storage stability, and prospects for reducing antigenicity of whey proteins for application in the food industry. MpuAP hydrolyzed primarily five peptide bonds, Gln⁴–His⁵, His¹⁰–Leu¹¹, Ala¹⁴–Leu¹⁵, Gly²³–Phe²⁴ and Phe²⁴–Phe²⁵ in the oxidized insulin B-chain. The lyophilized form of the enzyme was well preserved at 30–40°C for 7 days without stabilizers. To investigate the possibility of reducing the antigenicity of the milk whey protein, enzymatic hydrolysates of the whey protein were evaluated by inhibition ELISA. Out of the three main components of whey protein, casein and α-lactalbumin were efficiently degraded by MpuAP. The sequential reaction of MpuAP and trypsin against the whey protein successfully degraded casein, α-lactalbumin and β-lactoglobulin with the highest degree of hydrolysis. As a result, the hydrolysates obtained by using the MpuAP–trypsin combination showed the lowest antigenicity compared with the single application of pepsin, trypsin or pepsin–trypsin combination. Therefore, the overall result suggested that the storage-stable MpuAP and trypsin combination will be a productive approach for making hypoallergic bovine milk whey protein hydrolysates.     

Purification and characterization of the fusion protein trypsin-streptavidin expressed in Escherichia coli

Expression of fusion protein trypsin-streptavidin (TRYPSA)⁴ in Escherichia coli was evaluated and the protein purified. Protein expression was induced by 1 mM isopropylthio--D-galactoside (IPTG), and the enzyme activity was measured by the hydrolysis rate of p-toluenesulfonyl-l-arginine methyl ester (TAME). Expression of the fusion protein in the cell-free extract decreased with increased induction time; correspondingly, that in the inclusion bodies increased. The total expression in Luria–Bertani broth (LB) and Terrific Broth (TB) media reached the highest levels in 2 hr at 30°C. The optimum expression level was 35 and 48 U/L in LB and TB, respectively. Expression of the fusion protein was verified by Western Blot analysis using streptavidin antiserum, and the fusion protein was purified using a benzamidine Sepharose 6B affinity column at room temperature. The molecular size of the soluble purified fusion protein was determined by size-exclusion chromatography using Superose 12 FPLC. A molecular weight of 39–40 kDa was obtained, indicating that the soluble protein exists as a monomer; thus, the presence of the trypsin domain must prevent the streptavidin domain from tetramer formation.     

Glass-immobilized glycated-trypsin: A novel modified trypsin that is remarkably thermostable

Porcine trypsin was glycated with glucose and covalently immobilized through its carboxyl groups onto aminated glass beads to produce porcine immobilized glycated-trypsin (IGT). On incubation at 60 °C and pH 8, IGT retained its full activity for 8 h and 50% of its activity after 24 h. In comparison, under the same conditions porcine native trypsin lost 80% of its activity in 2 h and was completely inactivated in less than 4 h. The rate of autolysis of porcine glycated-trypsin at 37 °C was 40% that of native trypsin and with IGT there was no significant autolysis, even at elevated temperatures as high as 60 °C. Glycation significantly increased the stability of trypsin and immobilization also significantly increased the stability of trypsin. The remarkable thermostability of IGT is attributed to a synergistic effect when these two modifications are combined. Tryptic fragmentation of denatured proteins with IGT can be performed at 60 °C for shorter digestion times and with smaller amounts of enzyme than normally employed to achieve complete digestion with soluble forms of trypsin. Prior denaturation of proteins for tryptic digestion is not required with IGT as in situ denaturation and digestion can be achieved simultaneously at 60 °C with an enzyme:protein mass ratio as low as 1:1000.     

Buffalo Cheese Whey Proteins,Identification of a 24 kDa Protein and Characterization of Their Hydrolysates: In Vitro Gastrointestinal Digestion

Milk whey proteins are well known for their high biological value and versatile functional properties, characteristics that allow its wide use in the food and pharmaceutical industries. In this work, a 24 kDa protein from buffalo cheese whey was analyzed by mass spectrometry and presented homology with Bos taurus beta-lactoglobulin. In addition, the proteins present in buffalo cheese whey were hydrolyzed with pepsin and with different combinations of trypsin, chymotrypsin and carboxypeptidase-A. When the TNBS method was used the obtained hydrolysates presented DH of 55 and 62% for H1 and H2, respectively. Otherwise for the OPA method the DH was 27 and 43% for H1 and H2, respectively. The total antioxidant activities of the H1 and H2 samples with and without previous enzymatic hydrolysis, determined by DPPH using diphenyl-p-picrylhydrazyl radical, was 4.9 and 12 mM of Trolox equivalents (TE) for H2 and H2Dint, respectively. The increased concentrations for H1 and H2 samples were approximately 99% and 75%, respectively. The in vitro gastrointestinal digestion efficiency for the samples that were first hydrolyzed was higher compared with samples not submitted to previous hydrolysis. After in vitro gastrointestinal digestion, several amino acids were released in higher concentrations, and most of which were essential amino acids. These results suggest that buffalo cheese whey is a better source of bioavailable amino acids than bovine cheese whey.