Affinity chromatography of Ankistrodesmus braunii nitrate reductase using blue dextran-sepharose (author's transl)

Blue Dextran has been coupled covalently to Sepharose-4B to purify the enzymatic complex NAD(P)H-nitrate reductase (EC 1.6.6.2) from the green alga Ankistrodesmus braunii by affinity chromatography. The optimum conditions for the accomplishment of the chromatographic process have been determined. The adsorption of nitrate reductase on Blue Dextran Sepharose is optimum when a phosphate buffer of low ionic strength and pH 6.5-7.0 is used. Once the enzyme has been bound to Blue Dextran Sepharose, it can be specifically eluted by addition of NADH and FAD to the washing buffer. However, none of the nucleotides added separately is able to promote the elution of the enzyme from the column. The elution can be also achieved, but not specifically, by increasing the ionic strength of the buffer with KCl. These results have made possible a procedure for the purification of A. braunii nitrate reductase which led to electrophoretic homogeneity, with an overall yield of 70% and a specific activity of 49 units/mg of protein.     

Soluble expression and purification of porcine pepsinogen from Pichia pastoris

This paper presents a new system for the soluble expression and characterization of porcine pepsinogen from the methylotrophic yeast Pichia pastoris. The cDNA that encodes the zymogenic form of porcine pepsin (EC 3.4.23.1) was cloned into the EcoRI site of the vector pHIL-S1 downstream from the AOX1 alcohol oxidase promoter. After P. pastoris transformation, colonies were screened for expression of pepsinogen based on enzyme activity of the active form, pepsin. The recombinant enzyme was purified 138-fold by anion exchange and affinity column chromatography. Homogeneity was confirmed through SDS-PAGE, Western blot, and N-terminal sequencing. When compared to commercial pepsin, the recombinant pepsin had similar kinetic profiles, pH/temperature stability, and secondary/tertiary conformation. A glycosylated form was also isolated and found to exhibit kinetic and structural characteristics similar to those of the commercial and wild-type pepsin, but was slightly more thermal stable. The above results indicate that the P. pastoris expression system offers a convenient and efficient means to produce and purify a soluble form of pepsin(ogen).     

Pepsinogen C and pepsin C: Further purification and amino acid composition

Pepsinogen C and pepsin C from the pig have been further purified by chromatography on DEAE-cellulose and by exclusion chromatography and the specific activities (with haemoglobin substrate) found are higher than those previously reported. The final preparations are homogeneous on electrophoresis in starch gel at three pH values except for contamination with less than 4% of pepsinogen and pepsin respectively. Pepsinogen C, like pepsin C, contains no phosphate. The amino acid compositions show some marked differences from those of pepsinogen and pepsin especially in the content of basic amino acids, glutamic acid, aspartic acid, leucine and isoleucine. The molecular weights of the enzyme and zymogen, obtained from the amino acid compositions, are 41400 and 36000 respectively, similar to those of pepsinogen and pepsin.     

Molecular characteristics of pepsinogen and pepsin from duck glandular stomach

1. Two procedures were developed for the preparation of duck pepsinogen, an enzyme from the family of aspartic proteases (EC 3.4.23.1) and its zymogen. 2. The amino acid composition, sugar content and the partial N- and C-terminal sequences of both the enzyme and the zymogen were determined. These sequences are highly homologous with the terminal sequences of chicken pepsin(ogen). 3. Duck pepsinogen and pepsin are unlike other pepsin(ogen)s in being relatively stable in alkaline media: pepsinogen is inactivated at pH 12.1, pepsin at pH 9.6. 4. Duck pepsin is inhibited by diazoacetyl-D,L-norleucine methyl ester (DAN), 1,2-epoxy-3(p-nitrophe-noxy)propane (EPNP), pepstatin and a synthetic pepsin inhibitor Val-D-Leu-Pro-Phe-Phe-Val-D- Leu. The pH-optimum of duck pepsin determined in the presence of synthetic substrate is pH 4. 5. Duck pepsin has a marked milk-clotting activity whereas its proteolytic activity is lower than that of chicken pepsin. 6. The activation of duck pepsinogen is paralleled by two conformational changes. The activation half-life determined in the presence of a synthetic substrate at pH 2 and 14 degrees C is 20 sec.     

Immobilized-metal-ion affinity chromatography as a tool for the qualitative study of pepsinogen phosphorylation.

Affinity chromatography on immobilized Fe(3+) ions--immobilized-metal-ion affinity chromatography (IMAC) method--was used for the determination of pepsin and pepsinogen phosphorylation. IMAC is a very powerful method for detailed studies of proteins. Dephosphorylation of the pepsinogens and pepsins has no effect on their proteolytic ability. For this reason, the determination of proteolytic activity was used for the detection of pepsinogen (pepsin) presence in the collected fractions as a very suitable and specific method. Pepsins and their zymogens probably have the same amounts of phosphate ions in their molecule. The exact definition of conditions is very important for the prepurification of the proteinases and for their analysis.     

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.     

Mechanism of intramolecular activation of pepsinogen. Evidence for an intermediate delta and the involvement of the active site of pepsin in the intramolecular activation of pepsinogen.

Intramolecular pepsinogen activation is inhibited either by pepstatin, a potent pepsin inhibitor, or by purified globin from hemoglobin, a good pepsin substrate. Also, pepsinogen at pH 2 can be bound to a pepstatin-Sepharose column and recovered as native zymogen upon elution in pH 8 buffer. Kinetic studies of the globin inhibition of pepsinogen activation show that globin binds to a pepsinogen intermediate. This interaction gives rise to competitive inhibition of intramolecular pepsinogen activation. The evidence presented in this paper suggests that pepsinogen is converted rapidly upon acidification to the pepsinogen intermediate delta. In the absence of an inhibitor, the intermediate undergoes conformational change to bind the activation peptide portion of this same pepsinogen molecule in the active center to form an intramolecular enzyme-substrate complex (intermediate theta). This is followed by the intramolecular hydrolysis of the peptide bond between residues 44 and 45 of the pepsinogen molecule and the dissociation of the activation peptide from the pepsin. Intermediate delta apparently does not activate another pepsinogen molecule via an intermolecular process. Neither does intermediate delta hydrolyze globin substrate.     

Aromatic amino acids and their derivatives as ligands for the isolation of aspartic proteinases

Affinity chromatography was used to study an interaction of aspartic proteinases with immobilized aromatic amino acids and their derivatives. The following ligands were used: L-tyrosine, 3-iodo-L-tyrosine, 3,5-diiodo-L-tyrosine, L-phenylalanine, p-iodo-L-phenylalanine and N-acetyl-L-phenylalanine. With the exception of the last one, ligands were coupled directly to divinyl sulfone activated Sepharose 4B. For the preparation of immobilized N-acetyl-L-phenylalanine, divinyl sulfone activated Sepharose 4-B with linked ethylene diamine was used. Porcine pepsin was used for the evaluation of the capacity of the prepared affinity carriers. The capacity of the immobilized amino acid derivatives significantly increased in comparison with the non-derivatized amino acids. The prepared immobilized ligands were further used for the separation of human pepsinogens.     

The purification and properties of a single chicken pepsinogen fraction and the pepsin derived from it

1. Evidence is given for the presence of at least five pepsinogens in a crude extract of mixed chicken stomachs. One of these was purified and could be activated to yield a single pepsin. 2. The molecular weights of the pepsinogen and pepsin were 36000 and 34000 respectively. The pepsin associated at low pH values and low ionic strength. 3. The amino acid analyses of both proteins are given. The pepsin was devoid of phosphate but contained carbohydrate. 4. The N-terminal amino acids of pepsinogen and pepsin were serine and threonine respectively. Five amino acids were released by carboxypeptidase A and it was deduced that serine may be the C-terminal one. 5. Each protein contained one thiol group per molecule as determined by titration with p-chloromercuribenzoate. The rate of the reaction was very rapid with pepsin, but much slower with pepsinogen, although the same group appeared to react in both instances. The enzymic activity of pepsin was unaffected by the modification. 6. The isoionic point of the pepsin was close to pH4.0 and the enzyme was stable for long periods at pH values up to 7.0. 7. The enzyme hydrolysed bisphenyl sulphite almost as rapidly as did pig pepsin A.     

N-terminal portion acts as an initiator of the inactivation of pepsin at neutral pH.

Porcine pepsin, an aspartic protease, is unstable at neutral pHs where it rapidly loses activity, however, its zymogen, pepsinogen, is stable at neutral pHs. The difference between the two is the presence of the prosegment in pepsinogen. In this study, possible factors responsible for instability were investigated and included: (i) the distribution of positively charged residues on the surface, (ii) an insertion of a peptide in the C-terminal domain and (iii) the dissociation of the N-terminal fragment of pepsin. Mutations to change the number and the distribution of positive charges on the surface had a minor effect on stability. No effect on stability was observed for the deletion of a peptide from the C-terminal domain. However, mutations on the N-terminal fragment had a major impact on stability. At pH 7.0, the N-fragment mutant was inactivated 5.8 times slower than the wild-type. The introduction of a disulfide bond between the N-terminal fragment and the enzyme body prevented the enzyme from denaturing. The above results showed that the inactivation of pepsin was initiated by the dissociation of the N-fragment and that the sequence of this portion was a major determinant for enzyme stability. Through this study, we have created porcine pepsin with increased pH stability at neutral pHs.     

Electrostatic and hydrophobic effects in affinity chromatography

A semi–quantitative theory is developed to explain the nonspecific binding of proteins to substituted affinity chromatography supports due to electrostatic and hydrophobic interactions. The equilibrium constant for the absorption of an enzyme to a solid support, and the rate of desorption of the enzyme are studied as functions of ionic strength. Experimental measurements were taken of the adsorption equilibrium constant and rate of desorption of E. coli β–galactosidase on Sepharose 4B substituted with 3, 3,-diaminodipropylamine in batch systems. It was found that the enzyme adsorption exhibits a hysteresis effect as the ionic strength is increased and then decreased. Furthermore, the adsorption of theenzyme becomes more reversible at the lower ionic strengths, while at the higher ionic strengths it is essentially irreversible. Using the measured equilibrium constants, and knowing the region of ionic strength where the adsorption becomes reversible, we were able to predict the desorption of enzyme in a continuous stirred tank as a function of time when a decreasing linear gradient of ionic strength was introduced into a slurry. It was found that the presence of another protein, hemoglobin, does not affect these results, and therefore can be separated from the enzyme.     

Method of purification of glucose oxidase by means of affinity chromatography on immunoabsorbent]

The possibility to purify glucose oxidase from Penicillium vitale on immunosorbent containing specific antibodies to the enzyme covalently bound with Sepharose 4B is studied. The method of affinity chromatography was applied, beside routine methods of fractionating blood serum proteins, to isolate specific antibodies from antiserum of rabbits immunized with glucose oxidase. Immobilized on Sepharose glucose oxidase was used as biospecific sorbent. Specific antibodies to the enzyme were isolated using chromatograpy of gamma-globulins mixture followed by protein desorption from the column with 1 M NaC1 and 3% glucose. Antibodies were immobilized by their covalent binding to activated Sepharose. The immunosorbent obtained was used to purify low active preparation of glucose oxidase by means of affinity chromatography under conditions worked out for the antibodies isolation. The enzyme was eluted from the column with 1 M NaC1 (pH 3.0) containing 3% glucose. 5-Fold purified enzyme preparation was isolated.     

Purificaton and characterization of a pepsinogen and its pepsin from proventriculus of the Japanese quail

A crude extract of the proventriculus of the Japanese quail gave at least five bands of peptic activity at pH 2.2 on polyacrylamide gel electrophoresis. The main component, constituting about 40% of the total acid protease activity, was purified to homogeneity by hydroxyapatite and DEAE-Sepharose column chromatographies. At below pH 4.0, the pepsinogen was converted to a pepsin, which had the same electrophoretic mobility as one of the five bands of peptic activity present in the crude extract. The molecular weights of the pepsinogen and the pepsin were 40 000 and 36 000, respectively. Quail pepsin was stable in alkali up to pH 8.5. The optimal pH of the pepsin on hemoglobin was pH 3.0. The pepsin had about half the milk-clotting activity of purified porcine pepsin, but the pepsinogen itself had no activity. The hydrolytic activity of quail pepsin on N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine was about 1% of that of porcine pepsin. Among the various protease inhibitors tested, only pepstatin inhibited the proteolytic activity of the pepsin. The amino acid composition of quail pepsinogen was found to be rather similar to that of chick pepsinogen C, and these two pepsinogens possessed common antigenicity.     

Interaction of pepstatin with pig pepsinogen.

In contrast with pepsin, pepsinogen does not bind pepstatin at pH values between 5.3 and 2.5. Pepsinogen is not retarded by pepstatin immobilized on to aminohexyl-Sepharose at pH5.3 or 4.1, whereas at pH3.0 activation takes place during the chromatography, with retardation of the resultant pepsin.     

Predicting the elution behavior of proteins in affinity chromatography on non-porous particles.

Affinity chromatography on non-porous particles of microsize is particularly useful for the rapid analysis and micropreparative separation of proteins. The elution behavior of proteins in an affinity column packed with non-porous copolymerized particles of styrene, methyl methacrylate and glycidyl methacrylate was investigated both theoretically and experimentally, using the lysozyme-Cibacron Blue 3G-A affinity system. Equations used to predict the elution profiles, resulting from the elution by increasing the ionic strength (NaCl concentration) in the mobile phase, were obtained. The maximum adsorbate concentration, desorption rate constant and equilibrium constant under elution conditions were determined by matching experimental data with predicted elution profiles. Based on the parameters determined at a flow-rate of 0.5 ml/min and with 1 M NaCl in the elution buffer, the model equations could predict the elution profiles for other experimental runs, where different flow-rates and sodium chloride concentrations were used. Both the experimental and predicted results revealed that the affinity interaction kinetics are not significantly influenced by the flow-rate and, hence, the film mass transfer. To elute bound lysozyme from immobilized dye ligand, a higher value of the ionic strength leads to a faster elution and a sharper elution peak. The influence of elution conditions on the kinetic and thermodynamic parameters and, consequently, on the elution peak profiles was evaluated. The model equations can also predict the behavior of protein elution from an affinity column by changing the pH of the mobile phase, according to a previous study.     

Activation mechanism of monkey and porcine pepsinogens A. One-step and stepwise activation pathways and their relation to intramolecular and intermolecular reactions

The activation of Sepharose-bound monkey pepsinogen A under acidic conditions proceeded by cleavage of the Leu47-Ile48 bond, indicating the occurrence of the intramolecular one-step activation, although the rate of cleavage was very slow. On the other hand the activation of monkey pepsinogen A in solution was highly dependent on pepsinogen concentration and the addition of exogenous pepsin A accelerated the rate of activation, indicating the predominance of intermolecular reaction. The cleavage site, however, was also restricted to the Leu47-Ile48 bond. Thus, apparently exclusive one-step activation occurred in monkey pepsinogen. The activation of porcine pepsinogen A in solution was also dependent on pepsinogen concentration and the addition of exogenous pepsin A accelerated the rate of activation. The major cleavage site by the exogenously added pepsin was the Leu44-Ile45 bond. Therefore the site most susceptible to the intermolecular attacks was the bond connecting the activation segment and the pepsin moiety in both monkey and porcine pepsinogens. In porcine pepsinogen, however, a part of the zymogen was activated through the intermediate form, and an intramolecular reaction was suggested to be involved in the generation of this form. These results showed that in both pepsinogens A the intramolecular reaction occurred, first yielding pepsin A or the intermediate form, which then acted intermolecularly on the remaining pepsinogen or the intermediate form to complete the activation in a short time. A molecular mechanism for the activation reaction was proposed to explain consistently the experimental results.     

Reversed-phase high-performance liquid chromatography of peptides of porcine pepsin prepared by the use of various forms of immobilized α-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 α-chymotrypsin. Porcine pepsin was hydrolysed with soluble α-chymotrypsin, with α-chymotrypsins glycosylated with lactose or galactose coupled to hydrazide derivative of cellulose, with α-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 α-chymotrypsins immobilized by different methods. Best results were achieved after hydrolysis with α-chymotrypsin immobilized on poly(acrylamide-allyl glycoside) copolymers. α-Chymotrypsin immobilized by this way has further three times higher relative specific activity in comparison with the soluble one. Modified α-chymotrypsin was not suitable for efficient pepsin cleavage.     

Synthesis, purification, and active site mutagenesis of recombinant porcine pepsinogen

In order to carry out studies on structure and function relationships of porcine pepsinogen using site-directed mutagenesis approaches, the cDNA of this zymogen was cloned, sequenced, expressed in Escherichia coli, and the protein refolded, and purified to homogeneity. Porcine pepsinogen cDNA, obtained from a lambda gt10 cDNA library of porcine stomach contains 1364 base pairs. It contains leader, pro, and pepsin regions of 14, 44, and 326 residues, respectively. In addition, it also contains 5'- and 3'-untranslated regions. Four differences are present between the sequence deduced from the cDNA and the pepsinogen sequence determined previously by protein chemistry methods. Residues P19 (in the pro region) and 263 are asparagines in the cDNA sequence instead of aspartic acids. Isoleucine 230 is not present in the cDNA sequence and residue 242 is a tyrosine in the cDNA instead of an aspartic acid. Porcine pepsinogen cDNA was placed under the control of a tac promoter in a plasmid and expressed in E. coli. The synthesis of pepsinogen was optimized to about 50 mg/liter of culture. The recombinant (r-) pepsinogen, which was insoluble, was recovered by centrifugation, washed, dissolved in 6 M urea in Tris-HCl, pH 8, and refolded by rapid dilution. r-pepsinogen was purified to homogeneity after chromatography on Sephacryl S-300 and fast protein liquid chromatography on a monoQ column. r-pepsinogen contains an additional methionine residue at the NH2 terminus as compared to native (n-) pepsinogen. However, r- and n-pepsinogens are indistinguishable in their intramolecular activation constants. After activation, r- and n-pepsins have the same NH2-terminal sequences as well as Km values. Based on these data, r-pepsinogen was judged suitable for mutagenesis studies. A mutant pepsinogen (D32A) with the active site aspartic acid changed to an alanine was produced and purified. D32A-pepsinogen did not convert to pepsin in acid solution but it bound to pepstatin with an apparent KD of about 5 x 10(-10) M. D32A-pepsinogen possesses no detectable proteolytic activity. These results indicate that (i) intramolecular pepsinogen activation is accomplished by the pepsin active site, and (ii) unlike subtilisin (Carter, P., and Wells, J. A. (1988) Nature 332, 564-568), the active site mutant of pepsin is not enzymically active.     

Pepsin immobilized on inorganic supports for the continuous coagulation of skim milk.

The milk-clotting enzyme pepsin was immobilized onto beads of alumina, titania, glass, stainless steel, iron oxide, and Teflon for treating skim milk in a fluidized-bed reactor. Two covalent attachment procedures using silanized supports and glutaraldehyde and two adsorption procedures were evaluated. The three best catalysts were titania and glass, using the covalent attachment procedure, and alumina, using the adsorption procedure at pH 1.2. The pepsin adsorbed on alumina catalyst has commercial potential compared to the previously used glass catalyst. Attempts to increase the stability of pepsin adsorbed on alumina by cross-linking with glutaraldehyde were unsuccessful owing to the low pH necessary for optimum pepsin adsorption; Desorption of pepsin from alumina during reactor operation was determined. Regeneration of spent catalysts was only partially successful.     

Isolation and characterization of a pepsin C zymogen produced by human breast tissues.

An aspartic proteinase present in cyst fluid from women with gross cystic breast disease was purified by a procedure involving affinity chromatography on pepstatin-agarose and size-exclusion high performance liquid chromatography. The amino-terminal sequence of the purified breast proteinase was identical to that corresponding to gastric pepsinogen C. Additional data on cleavage specificity, pH optimum, and immunological properties supported the close relationship between both molecules. Northern blot analysis and polymerase chain reaction amplification studies performed on RNAs obtained from normal and pathological breast tissues demonstrated that the protein is produced by mammary carcinomas and cysts, but not by the normal resting mammary gland. Immunohistochemical staining of paraffin-embedded tissue sections confirmed the existence of a subset of tumors that have the ability to synthesize and secrete this pepsin zymogen. On the basis of these results, we suggest that pepsinogen C expression by human mammary epithelium may be involved in the development of breast diseases, being also of potential interest as a biochemical marker of the hormonal imbalance underlying these pathologies.