Chicken Pepsin Research Articles

Chromophoric peptide substrates for activity determination of animal aspartic proteinases in the presence of their zymogens: A novel assay

Solid-phase synthesis was used for the preparation of pyroglutamyl-histidyl- p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide ( I) and glycyl-glycyl-histidyl- p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide ( II), two water-soluble and sensitive chromophoric substrates of chicken pepsin, hog pepsin A, and bovine spleen cathepsin D. The kinetic constants of hydrolysis of the p-nitrophenylalanyl-phenylalanyl bond of the substrates were measured by difference spectrophotometry at 308 nm (Δϵ = 860 m −1 cm −1) and by ninhydrin colorimetry (substrate I, ϵ 570 = 2.31 × 10 4 m −1 cm −1). The pH optimum of cleavage is 5 for the pepsins and 3.7 for cathepsin D. Since all three proteinases still have a significant activity at pH 5.5–6 a new, simple assay was designed for submicrogram quantities of pepsins in the presence of pepsinogens without interference of the latter. The method is particularly suitable for the analyses of the zymogen activation mixtures.

Kinetics of Thermal Inactivation of Chicken Pepsin

Thermal inactivation of chicken pepsin was studied at pH 3, 4, and 6. Application of heat to enzyme solutions in capillary tubes enabled attainment of short come-up times. Enzyme denaturation followed first order reaction kinetics, exhibiting reasonably high thermal resistance. Activation energies of denaturation were 25.6, 28.0, and 30.0 kcal/mole for pH 6, 4, and 3, respectively. These are considerably lower than corresponding activation energies for other commonly utilized milk clotting enzymes. Thermal resistance and low activation energy of denaturation of chicken pepsin calls for care to inactivate properly the residual enzyme of whey products where its proteolytic activity may be undesirable.

Combined Temperature-Concentration Effects on the Clotting Rate of Chicken Pepsin

Milk clotting rates with chicken pepsin were measured over a wide temperature range and at various enzyme concentrations, yielding isothermally linear relationship between the inverse of the clotting time and enzyme concentration. Two distinct approaches are shown with which the concentration-temperature-coagulation rate data set can be formulated into single overall mathematical equations. One approach uses an Arrhenius type format, whereas in the other, a direct temperature relationship is employed in a manner analogous to the way that thermal resistance of microorganisms is expressed. The overall rate equations enable accurate prediction of clotting times under given conditions of enzyme concentration and temperature and conceivably can be extended to include additional independent variables.

Amino acid sequence around the reactive cysteinyl residue of chicken pepsin

The amino acid sequence of the cysteine-containing peptide was determined in two different manners. 1) Chicken pepsin was covalently coupled via its SH-group to Thiopropyl-Sepharose 6B, subjected to peptic digestion and cysteinyl peptides, generated by multiplicity of peptic attack on the sequence ..-Tyr-Tyr-Cys-Asn-Phe-.. (marked by arrows), were eluted by 2-mercaptoethanol. 2) Systematic structure analysis of the tryptic-chymotryptic digest of chicken pepsinogen afforded peptide Tyr-Tyr-Cys-Asn-Phe-Asp-Gly-Ile-Leu-Gly-Leu, whose C-terminal part is highly homologous with other pepsins. These data permitted the cysteinyl residue to be assigned to position 115 (numbered according to hog pepsin A) in a relatively variable region. The same position is occupied by an alanyl residue in hog pepsin A and calf chymosin; this replacement can be considered conservative from the viewpoint of molecular volume.

HPLC Separation of Bitter Peptides from Cheddar Cheese

High performance liquid chromatography was used to separate bitter peptides extracted from Cheddar cheese coagulated with chicken pepsin. A reversed phase octadecylsilane type column coupled with linear gradient elution from water to 91% methanol enabled the bitter extract to be separated into at least seventy-one compounds shown to be peptides. Gel filtration was also used but the one bitter fraction, as determined by sensory testings, obtained in this manner was separated into at least forty-four different components by high performance liquid chromatography. The chromatograms from both methods were divided into bitter and nonbitter fractions, each containing numerous peaks, according to sensory analysis. The average molecular weight of the bitter fraction obtained by gel filtration was established to be approximately 190 daltons. Ultracentrifugation of the high performance liquid chromatography fractions supported this estimate. Fractions collected by both separation techniques were subjected to sensory and chemical analyses. The two bitter high performance liquid chromatography fractions had slightly higher average hydrophobicity values than the nonbitter fractions, but this did not hold for the bitter fraction obtained by gel chromatography. Valine and leucine occurred at a higher level in the high performance liquid chromatography separated bitter fractions than in the nonbitter fractions, while lysine values were elevated in the bitter fraction separated by gel chromatography. There was an increase in retention time with increasing average hydrophobicity of the bitter extract fractions, but no such trend appeared for the pure proteins and peptides tested; there was, however, an apparent positive relationship between retention time and molecular weight.

Characterization of the sulfated glycopeptide of chicken pepsinogen

S-sulfonated chicken pepsinogen was digested with TPCK-trypsin; large tryptic peptides, separated on Sephadex G-25 fine, were subjected to additional cleavage with α-chymotrypsin. The hold-up fraction of the chymotryptic digest from the Sephadex G-25 column, was resolved by high voltage electrophoresis. The three most acidic zones contained glycopeptides of identical amino acid sequence Val-Ser-Thr-Asn-Glu-Thr-Val-Tyr, yet differed in the composition of the sugar moiety. These glycopeptides, moreover, bear different numbers of sulfate groups which enabled the resolution of the peptides. The most acidic glycopeptide contains 7 glucosamine residues, 3 mannose residues and 5 sulfate groups, the second one 6 glucosamine residues, 3 mannose residues and 4 sulfate groups and the slowest, minority glycopeptide, 5 glucosamine residues, 2 mannose residues and 2 sulfate groups. The entire sugar moiety is attached to one of the chain viaasparagine. In other experiments the glycopeptides were also isolated from the thermolytic digest of chicken pepsin; their C-terminal sequence was shorter by two amino acid residues. The tentative assignment of the glycopeptides to the amino acid sequence of pepsinogen resulted from the analysis of the limited tryptic digest of the whole protein molecule. Chicken pepsinogen is glycosylated at the site of the chain occupied by a phosphoserine residue in hog pepsinogen A.

Immobilization of proteins as a tool for studying primary structure around their cysteinyl residues

The primary structure around the single cysteinyl residue of chicken pepsin was investigated by binding the protein via this residue to an insoluble carrier. Carriers stable towards reagents used for the fragmentation of proteins and sequence analysis were prepared by coupling a spacer arm to polyN-hydroxymethyl acrylamide using a thioether bond that is potentially cleavable by mercuric ions (1). Phenacyl bromide group, attached to the free end of the spacer, reacted rapidly and specifically with the cysteinyl residue of chicken pepsin. Up to 300 mg of the enzyme were bound to 1 g of carrier.The polymer-bound protein was cleaved by trypsin or by cyanogen bromide or by a sequence of both. Fragments of 40-120 amino acid residues, depending on the method of cleavage, remained attached to the polymer through the cysteinyl residue. The compositions and partial sequences of these fragments revealed that the cysteinyl residue is located within or in the vicinity of a loop in the molecule formed by a disulfide bond.

Thermolytic digest of chicken pepsin

Chicken pepsin prepared by the activation of pepsinogen was digested with thermolysin. The thermolytic digest was fractionated by chromatography on Sephadex G-25 fine. Certain fractions were subsequently subjected to ion exchange chromatography on Dowex 50-X2. The final purification was effected by paper chromatography and high voltage electrophoresis. By these procedures a series of homogeneous peptides was obtained; of the latter 54 nonoverlapping (save for a few exceptions) peptides are described in this paper. These peptides in addition to the thermolytic peptides reported before represent 80% of the linear structure of the whole molecule. The N-terminal amino acid sequence of chicken pepsin is discussed from the viewpoint of the recent data obtained by the analysis of the thermolytic digest.

Cheddar Cheese Made with Chicken Pepsin

Commercial chicken pepsin was compared to calf rennet and a mixture of calf rennet and porcine pepsin as coagulating enzymes for the production of pilot plant quantities (25 kg) of Cheddar cheese. Yield data indicated that a significantly lower yield of cheese may be expected using chicken pepsin. Acid soluble nitrogen levels were considerably higher in the chicken pepsin cheese at both the 3 week and 3 month sampling periods, indicating a higher level of proteolysis with this enzyme. Instrumental and sensory texture analyses showed the chicken pepsin cheese lacked firmness compared to the other cheeses. The sensory panel also perceived a high and objectionable level of bitterness in cheese prepared with the chicken enzyme. Scanning electron microscopy revealed a more open, less compact protein network in chicken pepsin cheese that is probably responsible for its poorer texture. Electrophoresis of the respective cheese caseins showed a larger beta casein peak for calf rennet cheese. The alpha S peak was composed of four separate bands and their relative proportions were influenced by enzyme source. It is concluded that the chicken pepsin enzyme used in this study does not produce an acceptable Cheddar cheese either from quality or yield considerations. This is considered due to extensive proteolysis that led to defects in flavor and texture. A more highly purified enzyme preparation should be evaluated in the future.

Isolation and characterization of a peptide derived from the active site of chicken pepsin

A peptide was isolated from chicken pepsin which contains the aspartic acid residue reacting with diazoacetyl-D,L-norleucine methyl ester in the presence of Cu2+ -ions. The peptide is N-terminated with isoleucine and contains (besides isoleucine) valine, aspartic acid, two threonines, serine, and leucine. In concurrent experiments a peptide of the same composition was isolated from the thermolysin digest of chicken pepsin and its sequence determined as Ile-Val-Asp-Thr-Gly-Thr-Ser-Leu. Since both peptides have entirely identical amino acid composition and other characteristics, the sequenced peptide corresponds to the peptide isolated from the active site of the enzyme.

Primary structure of peptides which form the disulfide bonds of chicken pepsin

The molecule of chicken pepsin is cross-linked by three disulfide bonds. The structures of the half-cystine peptides which form these bonds were determined by the analysis of different enzymic digests of the enzyme. The order of the disulfide bonds in the molecule was elucidated with regard to sequential homologies between chicken pepsin and other acid proteases. The three disulfide bonds of chicken pepsin, numbered from the N-terminus of pepsin, are: 1st bond Ile-Tyr-Cys-(Lys-Ser-Ser-Ala)-Cys-Ser-Asn-His-Lys; 2nd bond Val-Ala-Cys-Cys-(Thr-Phe)-Gln-Ala; 3rd bond Asp-Leu-Gly-Val-Ser-Ser-Asp-Gly-Glu-Ile-Ser-Cys-(Asp-Asp-Ile-Ser-Lys-Leu-Pro-Asp)-Cys-(Ser-Gly-Asp-Glu-Asn-Leu-Val)-Met.

Isolation and some properties of an acid protease from Fasciola hepatica.

A new protease, detected in an extract of Fasciola hepatica, was isolated and partly purified. The pH optimum for the cleavage of denaturated haemoglobin by the enzyme is pH 3.0. This proteolytic activity is inhibited by diazoacetylnorleucine methyl ester, pepstatin, the pepsin inhibitor from Ascaris suum, and phenylalanine. The cathepsin D inhibitor from potatoes, EDTA, mercaptoethanol and the inorganic salts tested have no inhibitory effect. The cleavage of the B-chain of oxidized insulin by enzyme was studied and compared with the digestion of the same substrate by chicken and pig pepsin. The protease from Fasciola hepatica belongs to the carboxyl group of proteases and probably plays an important role in helminth nutrition.

Efficacy of Chicken Pepsin as a Milk Clotting Enzyme

Comparative laboratory tests of cheesemaking show similarity between chicken pepsin and calf rennet. Suitability of chicken pepsin for large-scale production of Emmental (Swiss) and Kashkaval-type cheeses was tested.

The first step in the activation of chicken pepsinogen is similar to that of prochymosin

Chicken pepsinogen was incubated at pH2.5 with pepstatin. The zymogen activated itself by a sequential mechanism and an intact peptide derived from residues 1-26 in the protein was released in the first step. This peptide was found to inhibit the milk-clotting activities of pig and chicken pepsins and calf chymosin but to different extents.

N-Terminal sequence analysis of chicken pepsinogen and pepsin

N-Terminal sequence analysis of chicken pepsinogen and pepsin

The effect of acid proteinase inhibitors on chicken pepsin.

1. The activity of chicken pepsin was partially inhibited by dimethyl-(2-hydroxy-5-nitrobenzyl)sulphonium bromide, but was unaffected by p-bromophenacyl bromide. 2. In the presence of Cu2+, diazoacetylnorleucine methyl ester completely inactivated chicken pepsin with the incorporation of 1 mol/mol. The mechanism of the reaction was similar to that with pig pepsin. 3. Chicken pepsin was completely inactivated by 2-diazo-4-bromoacetophenone in the presence of Cu2+. 4. Chicken pepsin was almost completely inactivated by 1,2-epoxy-3-(p-nitrophenoxy)propane at 25 degrees C, 3-4mol of inhibitor/mol being incorporated. The reaction at 10 degrees C was investigated briefly. 5. Calf chymosin was inactivated by 1,2-epoxy-3-(p-nitrophenoxy)propane at 10 degrees C, the incorporation of 1 mol/mol being required for complete inhibition. 6. The characteristics of the reactions of chicken pepsin with the above compounds were compared with those of other acid proteinases.

Modulation of the Enzymic Activity of Chicken Pepsin by the Covalent Modification of Its Single ‐ SH Group

The single cysteinyl residue of chicken pepsin was modified with a wide spectrum of reagents to produce mixed disulfides or alkylated derivatives. All these derivatives showed enhanced catalytic activity towards the synthetic peptide Z-Ala-Ala-Phe-OPrP, where OPrP is the 3-(4-pyridyl)-propyl-1-oxy group. The overall catalytic constant kcat/Km for these derivatives was 4 -- 25-fold larger than that of the native enzyme. The activity of the enzyme towards denatured hemoglobin was slightly decreased (10--45%) by these modifications. When the mixed disulfide derivatives were treated with excess mercaptan, the sulfhydryl group was regenerated and activity reverted to that of the native enzyme. The --SH group of chicken pepsin reacted preferentially with reagents containing an aromatic group. The reaction was found to depend on the ionization of a single group, presumably the --SH itself, with a pKa = 7.5. The rate of reaction of the fully deprotonated species with various disulfides was 100--1000-fold smaller than that of the --SH group of glutathione. It is suggested that the groups attached covalently to the sulfhydryl also interact with other amino acid side chains in the protein thereby affecting the active center of chicken pepsin.

ChemInform Abstract: ISOLATION AND SOME PROPERTIES OF CHICKEN PEPSIN

A novel method of isolation of chicken pepsins by chromatograph y on Sepharose 4B with a covalently attached mercury derivative is described. The chief advantages of this isolation procedure are a 6-fold increase of the specific activity of the preparation in one operation and minimum losses of active material. The preparation obtained was resolved into 4 individual forms by chromatography on DEAE-cellulos e. All forms showed the presence of N-terminal serine. They have the same specificity when tested with the B-chain of oxidized insulin as substrate; this specificity differs from that of hog pepsin. The molecule of chicken pepsin and its zymogen, unlike other animal pepsins so far known, has a free thiol group1'2. This thiol group is obviously located on the surface of the molecule and in native unmodified pepsin it can be not only titrated but also chemically modified3. We considered therefore worthwhile to use the presence of this free SH-group for the isolation of chicken pepsin by affinity chromatography. In this paper we report on the isolation of chicken pepsin on a mercurial Sepharose column and on the resolution of its individual forms and their characterization.

ChemInform Abstract: AFFINITY CHROMATOGRAPHY OF CHICKEN PEPSIN USING PEPSIN INHIBITOR FROM ASCARIS LUMBRICOIDES AS AFFINANT

Chemischer InformationsdienstVolume 6, Issue 19 Natural Products ChemInform Abstract: AFFINITY CHROMATOGRAPHY OF CHICKEN PEPSIN USING PEPSIN INHIBITOR FROM ASCARIS LUMBRICOIDES AS AFFINANT H. KEILOVA, H. KEILOVASearch for more papers by this authorA. SALVETOVA, A. SALVETOVASearch for more papers by this authorV. KOSTKA, V. KOSTKASearch for more papers by this author H. KEILOVA, H. KEILOVASearch for more papers by this authorA. SALVETOVA, A. SALVETOVASearch for more papers by this authorV. KOSTKA, V. KOSTKASearch for more papers by this author First published: May 13, 1975 https://doi.org/10.1002/chin.197519436AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume6, Issue19May 13, 1975 RelatedInformation

Affinity chromatography of chicken pepsin using pepsin inhibitor from Ascaris lumbricoides as affinant

Affinity chromatography of chicken pepsin using pepsin inhibitor from Ascaris lumbricoides as affinant