bluuebunny/crossref_metadata_2025_split huggingface dataset:
Substrate Stereochemistry of 3‐Hydroxy‐3‐Methylglutaryl‐Coenzyme A Synthase
Authors: Cornforth John Warcup , Phillips Gareth T. , Messner Burkhard , Eggerer Hermann 
Published: March 2005

Synthetic specimens ofR‐[2H1,3H1]acetate and ofS‐[2H1,3H1]acetate were converted into coenzyme A thiolesters.Each of these thiolesters was condensed with acetoacetyl‐CoA on 3‐hydroxy‐3‐methyl‐glutaryl‐CoA synthase.The products, which were found to be hydroxymethylglutaryl‐phosphopantetheines owing to a secondary cleavage of the coenzyme A moieties, were reduced chemically to 3Rmevalonates.Each mevalonate specimen, after mixing with [2‐14C]mevalonate, was converted into squalene and cholesterol by a rat liver preparation.Each cholesterol specimen was converted into androst‐1,4‐diene‐3,17‐dione by incubation with a strain ofMycobacterium phleiin the presence of an inhibitor.When the procedures 4 and 5 were carried out with authentic specimens of 2R‐ and 2S‐[2‐3H1]mevalonate, it was found that the androstadienedione had lost nearly all tritium derived from the 2S‐mevalonate but had retained nearly all tritium (relative to14C) derived from the 2R‐mevalonate.The androstadienedione derived, by procedures 1–5, fromR‐acetate showed a loss of more than half of its tritium (relative to14C). The androstadienedione derived by the same procedure fromS‐acetate lost less than half of its tritium.It is concluded that the reaction on hydroxymethylglutaryl‐CoA synthase is stereospecific and is associated with an intramolecular hydrogen isotope effect. If this effect is normal (kH/kD>1) then the condensation proceeds with inversion of configuration at the methyl group.
Secondary Isotope Effect in the Phosphorylase Reaction
Authors: Firsov Leonid M. , Bogacheva Tatjana I. , Bresler Simeon E. 
Published: March 2005

The mechanism of action of phosphorylase B was studied by means of both α deuterium and α‐tritium secondary kinetic isotope effects of the enzymatic reaction. Two pairs of labelled substrates were synthesized : [1‐3H]glucose 1‐phosphate, [U‐14C]glucose 1‐phosphate and [1‐14C, 1‐2H]glucose‐1‐phosphate with [6‐3H]glucose 1‐phosphate. The measurements showed that the isotope effects of the enzymatic synthesis of glycogen as well as of the reverse reaction of glycogen phosphorolysis were equal to zero.The secondary α‐tritium isotope effect of the acid hydrolysis of glucose‐1‐phosphate was estimated. The ratio KH/K3H in this case was found to be 1.21.These data are consistent with the mechanism of direct displacement at the C‐1 atom of glucose and the formation of an intermediate enzyme · glucose‐1‐phosphate complex depending on the presence of glycogen.
5′‐Ends of Ribosomal and Ribosomal Precursor RNAs from Xenopus laevis
Authors: Slack Jonathan M. W. , Loening Ulrich E. 
Published: March 2005

The 5′‐ends of 18‐S, 28‐S and 40‐S RNA from Xenopus laevis have been studied by two techniques. The first involves fractionation of alkaline hydrolysates on columns of DEAE‐Sephadex at pH 7.8 and 2.7, followed by thin‐layer chromatography on polyethyleneimine cellulose. The second involves labelling in vitro of the RNA species at the 5′‐end using polynucleotide kinase.The 40‐S molecule is shown not to bear a terminal 5′‐triphosphate. It has the 5′‐terminus guanosine 5′‐phosphate. Both the 18 and 28‐S RNAs have the 5′‐terminus uridine 5′‐phosphate. The 28‐S RNA has the 5′‐terminal sequence pU‐N‐N‐N‐Gp.These results are discussed in relation to the transcription of the ribosomal genes, the possible heterogeneity of the ribosomal precursor and the position of the 28‐S sequence within the precursor molecule.
28‐S RNA from Xenopus laevis Contains a Sequence of Three Adjacent 2′‐O‐Methylations
Authors: Slack Jonathan M. W. , Loening Ulrich E. 
Published: March 2005

Evidence is presented to show that an alkaline hydrolysis product from 40‐S and 28‐S RNA of Xenopus laevis is a tetranucleotide containing three adjacent 2′‐O‐methylations.This is the longest such sequence yet reported and is believed to be Gm‐Am‐Am‐Ap.
Transfer of Heme from Ferrihemoglobin and Ferrihemoglobin Isolated Chains to Hemopexin
Authors: Hrkal Zbyněk , Vodrážka Zdeněk , Kalousek Ivan 
Published: March 2005

Incubation of ferrihemoglobin as well as of hemoglobin isolated chains in ferri‐form with hemopexin at pH 7.0 leads to the gradual transfer of heme to the hemopexin molecules until the equilibrium is attained.The reaction of ferrihemoglobin with hemopexin can be described as a four‐stage process involving successive transfer of the four heme groups from hemoglobin tetramer to the four molecules of hemopexin. The heme binding sites of the hemoglobin molecule are nonequivalent and cooperative.The transfer of heme from the isolated hemoglobin chains in ferri‐form occurs as the simple competitive reaction which can be characterized by a single equilibrium constant.The affinity constant value for the binding of heme to the single binding center of hemopexin molecule was estimated as 1.9 × 1014× M−1.The direct transfer of heme from hemoglobin to hemopexin may occur in vivo as a part of hemoglobin degradation process.
Induction of Ethylmorphine Demethylation and Aniline p‐Hydroxylation by Various Catatoxic Steroids in Regenerating Rat Liver
Authors: Japundžić Ivanka , Japundžić Milorad , Szabo Sandor 
Published: March 2005

In rats, the administration of a single dose of 3β‐hydroxy‐20‐oxopregn‐5‐ene‐16α‐carbonitrile and 9α‐fluoro‐11β,‐17 ‐dihydroxy‐3‐oxo‐4‐androstene‐17α‐propionic acid, SC‐11927 at the time of partial hepatectomy or 20 h later, resulted in a 3 to 5‐fold increase in ethylmorphine demethylase and aniline hydroxylase activity in the regenerating liver. Using the inhibitors of protein synthesis dactinomycin and cycloheximide it was shown that the induction process in regenerating liver, as well as in the liver of sham‐operated controls, could be blocked at transcriptional and translational levels. The extent of induction of the same enzymes by 17‐hydroxy‐7α‐thioacetyl‐3‐oxo‐4‐androstene‐17α‐propionic acid γ‐lactone, spironolactone in regenerating liver was only one third of that in sham‐operated controls. Treatment with spironolactone for three days before operation did not increase the degree of enzyme induction in regenerating liver.Evidence is presented against a mutual exclusion of simultaneous cell proliferation and induction of mixed function oxidase. Both, liver growth rate and hydrolxyase activity can be simultaneously stimulated by catatoxic steroids. The possible occurrence of these two processes in different cell areas of the regenerating liver is discussed.
The Amino‐Acid Sequence of Porcine Adenylate Kinase from Skeletal Muscle
Authors: Heil Albert , Müller Gudrun , Noda Lafayette , Pinder Thomas , Schirmer Heiner , Schirmer Ilse , von Zabern Inge 
Published: March 2005

Adenylate kinase (ATP:AMP phosphotransferase) has been purified 490‐fold from porcine muscle with a final yield of 60 mg/kg muscle.
The amino‐acid composition is Asp11, Asn2, Thr14, Ser11, Glu19, Gln6, Pro6, Gly19, Ala8, Cys2, Val17, Met6, Ile9, Leu18. Tyr7, Phe5, Lys21, His2, Arg11.
The protein molecule is a single polypeptide chain of 194 amino‐acid residues with an acetyl‐methionine at the N‐terminus and a lysine residue at the C‐terminus.
Cyanogen bromide cleavage of carboxymethylated adenylate kinase yielded six fragments which were further degraded by using trypsin, chymotrypsin, thermolysin, subtilisin or α‐protease. Sequence data on the resulting peptides are summarized in the present report, full details are given in a supplementary paper which has been deposited at CNRS from where copies can be obtained.
The primary structure of porcine adenylate kinase is: Ac‐Met‐Glu‐Glu‐Lys‐Leu‐Lys‐Lys‐Ser‐Lys‐Ile10‐Ile‐Phe‐Val‐Val‐Gly‐Gly‐Pro‐Gly ‐ Ser‐Gly20‐Lys‐Gly‐Thr‐Gln‐Cys‐Glu‐Lys‐Ile‐Val‐Gln30‐Lys‐Tyr‐Gly‐Tyr‐Thr‐His‐Leu‐Ser‐Thr‐Gly40‐Asp‐Leu‐Leu‐Arg‐Ala‐Glu‐Val‐Ser‐Ser‐Gly50‐Ser‐Ala‐Arg‐Gly‐Lys‐Met‐Leu‐Ser‐Glu‐Ile60‐Met‐Glu‐Lys‐Gly‐Gln‐Leu‐Val‐Pro‐Leu‐Glu70‐Thr‐Val‐Leu‐Asp‐Met‐Leu‐Arg‐Asp‐Ala‐Met80‐Val‐Ala‐Lys‐Val‐Asp‐Thr‐Ser‐Lys‐Gly‐Phe90‐Leu‐Ile‐Asp‐Gly‐Tyr‐Pro‐Arg‐Glu‐Val‐Lys100‐Gln‐Gly‐Glu‐Glu‐Phe‐Glu‐Arg‐Lys‐Ile‐Gly110‐Gln‐Pro‐Thr‐Leu‐Leu‐Leu‐Tyr‐Val‐Asp120‐Ala‐Gly‐Pro‐Glu‐Thr‐Met‐Thr‐Lys‐Arg‐Leu‐Leu130‐Lys‐Arg‐Gly‐Glu‐Thr‐Ser‐Gly‐Arg‐Val‐Asp140‐Asp‐Asn‐Glu‐Glu‐Thr‐Ile‐Lys‐Lys‐Arg‐Leu150‐Glu‐Thr‐Tyr‐Tyr‐Lys‐Ala‐Thr‐Glu‐Pro‐Val160‐Ile‐Ala‐Phe‐Tyr‐Glu‐Lys‐Arg‐Gly‐Ile‐Val170‐Arg‐Lys‐Val‐Asn‐Ala‐Glu‐Gly‐Ser‐Val‐Asp180‐Asp‐Val‐Phe‐Ser‐Gln‐Val‐Cys‐Thr‐His‐Leu190‐Asp‐Thr‐Leu‐Lys.
Characterization of Blood‐Group‐H‐Active Ceramide Tetrasaccharide from Hog‐Stomach Mucosa
Authors: Slomiany Bronislaw L. , Slomiany Amalia , Horowtz Martin I. 
Published: March 2005

A fucose‐containing ceramide tetrasaccharide exhibiting blood‐group‐H activity was isolated from a lipid extract of hog stomach mucosa by column fractionations on DEAE‐cellulose and Florisil followed by purification on thin‐layer plates.By partial acid hydrolysis, sequential degradation with specific glycosidases and methylation analysis, the structure of this glycolipid was identified as: Fucα1 →, 2Galα1 → 3Galβ1 → 4Glc1 → 1Cer; where Fuc = fucose; Gal = glucose; Gal = glucose and Cer = ceramide.
Stepwise Dissociation of Histones from Rat‐Liver Chromatin in Alkaline Solutions
Authors: Russev Georgi , Venkov Christo , Tsanev Roumen 
Published: March 2005

Rat liver chromatin was sedimented in a sucrose density gradient under alkaline conditions. It is shown that in the pH interval 12.2—12.8 four histone species are dissociated stepwise in the sequence: F2b, F1, F2al and F3. Histone F2a2 remains bound to DNA even at pH 13.About 70% of the non‐histonc proteins dissociate from the chromatin at pH values lower than 12.2 and 30% remain bound to DNA in the range of pH studied.
Further Investigations on the Subunit Structure of Microsomal Carboxylesterases from Pig and Ox Livers
Authors: Junge Wolfgang , Krisch Klaus , Hollandt Heinrich 
Published: March 2005

Using sedimentation equilibrium studies, the molecular weight of pig liver esterase was found to be 163000. Sedimentation velocity experiments yielded a sedimentation coefficient s020,w of 7.9 S; the diffusion coefficient was found to be 40.7 μm2· s–1 by ultracentrifugation and 40.3 μm2· s–1 by analytical gel filtration. From these data a molecular weight of 181 000 was calculated. By analytical disc electrophoresis a molecular weight of 183000 was estimated.
Disc electrophoresis of ox liver esterase revealed three enzymatically active bands. The molecular weight of two of them was 54500 and that of the third 187000. Extrapolation of the sedimentation coefficients measured at very low enzyme concentrations to infinite dilution yielded a minimum value of S020,w= 4.0 S. Based on gel filtration results a diffusion coefficient of 66.0 μm2· s–1 was calculated. From these data a molecular weight of 57300 was obtained. Sedimentation equilibrium experiments of the ox enzyme showed a non‐linear plot of In absorbance versus r2 indicating heterogeneity with respect to molecular weight. The smallest species present had a molecular weight of 59000.
By sedimentation equilibrium experiments in the presence of 6 M guanidine‐HCl and 0.1 M 2‐mercaptoethanol, the subunit weight of pig liver esterase was found to be 58000. For the maleylated enzyme, values of 52500 (sedimentation equilibrium) and 56500 (from S20,w and D20,w) were obtained.
By covalent cross linking of the subunits with dimethyl suberimidate and subsequent polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, three bands of molecular weights 60000, 120000 and 180000 were found.

All these results give strong evidence for a three subunit structure of pig liver carboxylesterase.
Kinetic Properties of the Phosphofructokinase from Erythrocytes of Rats and Rabbits
Authors: Kühn Brigitte , Jacobasch Gisela , Gerth Christa , Rapoport Samuel M. 
Published: March 2005

The activity of the phosphofructokinase from erythrocytes of rats and rabbits, and presumably of the enzyme from other sources depend on the presence of either K+ or NH4+ ions. Their effects are not due to ionic strength. The apparent activation constant, KD, is about 1 mM for NH4+ and 10 mM for K+. While K+ ions appear to act only as activators of the catalytic site, NH4+ ions have a double action. They act both as activators and as positive effectors which abolish the cooperativity, presumably by affecting the interaction between the subunits. They increase the affinity of the enzyme for fructose 6‐phosphate and ATP as substrates, while the affinity for ATP as inhibitor is weakened. These effects are probably secondary to the changed interaction between the subunits. It is concluded that only data on the phosphofructokinase in K+ system are representative of normal intracellular conditions. In certain physiological and pathological states the effect of NH4+ ions on the allosteric properties of the enzyme is probably important.Pi is an activator of both the K+‐ and the NH4+‐activated enzyme, with an approximate KD of 1.6 mM. Its effect is therefore of importance under physiological and pathological conditions.The data on the purified K+‐activated enzyme are in good agreement with changes of the metabolic pattern of the intact erythrocyte.
Kinetic Properties of the Phosphofructokinase from Erythrocytes of Rats and Rabbits
Authors: Kühn Brigitte , Jacobasch Gisela , Gerth Christa , Rapoport Samuel M. 
Published: March 2005

The phosphofructokinase of the red cells of rats is exquisitely sensitive to inhibition by ATP. The apparent k1 for ATP is decreased with lower pH values and fructose‐6‐phosphate concentrations. 2,3‐Bisphophoglycerate, which is present in the red cells of man and many mammals, inhibits the phosphofructokinase in a synergistic manner with ATP. The inhibitory effect of phosphoenolpyruvate is biologically insignificant. It may be estimated that the enzyme would be practically completely inactive under cellular conditions without the action of positive effectors.There exist a multitude of positive effectors. Significant activations under cellular conditions are exercised by AMP, glucose 1,6‐bisphosphate and inorganic phosphate. Fructose 1,6‐bisphosphate activates to a limited extent and ADP only at alkaline pH values. All positive effectors produce a decreased inhibition by ATP and increased affinity of the enzyme for fructose 6‐phosphate. AMP is the activator with the highest affinity, while glucose 1,6‐bisphosphate may be considered as a baseline activator owing to its sluggish turnover.The data may be fitted in a qualitative manner to the model of Monod, Wyman and Changeux. It is possible that the negative anionic effectors act on the same site.
Inhibition of Horse‐Liver Alcohol Dehydrogenase by Pt(CN)42– and Au(CN)2–
Authors: Gunnarsson Per‐Olov , Pettersson Gösta , Zeppezauer Michael 
Published: March 2005

Single and multiple‐inhibition experiments provide evidence that the complex anion Pt(CN)42‐ is bound at a single site per subunit of liver alcohol dehydrogenase in strict competition with coenzyme, ADP‐ribose, AMP and (less than 100 mM) chloride ion, but independently of 1,10‐phenanthroline. Binding of adenosine decreases the stability constant for the enzyme · Pt(CN)42‐ complex from 16 mM‐1 to 4 mM‐1.Au(CN)2‐ is bound at two sites per subunit of the enzyme. Both interactions are competitive with coenzyme, ADP‐ribose and AMP, but independent of 1,10‐phenanthroline. Only one site is available for interaction with Au(CN)2‐ in the binary enzyme complexes formed with Pt(CN)42‐, chloride ion, or adenosine. Estimation of the corresponding binding constants for Au(CN)2‐ gave evidence for cooperativity between the two Au(CN)2‐ binding sites.These results strongly indicate that Pt(CN)42‐ is bound at a site within the protein region occupied by the phosphate groups of the coenzyme, and it is suggested that arginine‐47 in the enzymatic amino‐acid sequence is part of a common binding site for negative charges on the coenzyme and coenzyme competitive anions such as Pt(CN)42‐, Au(CN)2‐, chloride and iodoacetate. The second binding site for Au(CN)2‐ appears to be situated within the protein region occupied by the adenosine part of the coenzyme.
Purification and Properties of NADP‐Dependent 17β‐Hydroxysteroid Dehydrogenase Solubilized from Porcine‐Testicular Microsomal Fraction
Authors: Inano Hiroshi , Tamaoki Bun‐ichi 
Published: March 2005

17β‐Hydroxysteroid dehydrogenase, which catalyzes oxidoreduction between androstenedione and testosterone, has been solubilized by sonication from porcine testicular microsomal membrane for the first time. From the solubilized enzyme preparation, the dehydrogenase activity was precipitated by between 40 and 75% saturation of ammonium sulfate. After the precipitate was dissolved, the enzyme solution was applied to a Sephadex G‐100 column and the dehydrogenase activity emerged at 1.80 void volumes. For further purification, it was subjected to DEAE‐cellulose column chromatography; by eluting with a linear gradient of increasing phosphate concentration, the enzyme activity between 15 and 20 mM phosphate. Finally, when the partially purified enzyme preparation was applied to a Bio‐Gel P‐100 column, the dehydrogenase activity emerged at 1.46 void volumes. The final enzyme preparation was purified 1880‐fold in specific activity compared to the microsomal fraction.The purified enzyme preparation approached apparent homogeneity, checked by discontinuous polyacrylamide gel electrophoresis at pH 8.3 and by thin‐layer gel chromatography. The final preparation was able to produce 67.7 nmol testosterone from androstenedione per min per mg protein at 37°C in the presence of NADPH, and could be stored at–20°C for 6 months with no significant loss of the enzyme activity. The molecular weight and molecular radius of the 17β‐hydroxysteroid dehydrogenase was estimated to be about 35500 and 2.37 nm, respectively, from the results of the gel filtration chromatography. In the phosphate buffer pH 7.4 maximal rate of reduction of androstenedione by the enzyme was observed at 50°C, and Q10 for the enzyme was 3.7, while the activation energy was 19500 cal. The rate of reduction of androstenedione at 37.5°C was about a half of the maximal one at 50°C in the phosphate buffer, whereas the reduction rate at 37°C was higher than the one at 50°C in the citric acid‐Na2HPO4 buffer pH 7.3. The enzyme for androstenedione showed a pH optimum between 6.5 and 7.5 in the presence of NADPH at 37°C but the optimal pH of the enzyme was shifted to 8.5 by raising the incubation temperature to 50°C. Estrone was the acceptor of the hydrogen released from NADPH by the 17β‐hydroxysteroid dehydrogenase and dehydroepiandrosterone served a similar purpose for androstenedione. The oxidation of testosterone by the enzyme in the presence of NADP+ proceeded at the same rate as the reduction of androstenedione. The purified enzyme preparation was devoid of any alcohol or 3‐hydroxyhexobarbital dehydrogenase activities or other enzymes related to testicular steroidogenesis.
The Self‐Association of Chymotrypsinogen A
Authors: Tung Ming Sung , Steiner Robert Frank 
Published: March 2005

The self‐association of chymotrypsinogen A at pH 8.1 and at ionic strength of 0.021 has been examined by means of sedimentation equilibrium. The data have been analyzed by several methods and results compared. Application of the older methods available in the literature indicate that the concentration dependence of molecular weight may be well accounted for in terms of an indefinite association in which the consecutive stepwise association constants are equal and the second virial coefficient is small and positive.A new method has been developed, based on series expansion, which selects the values of the second virial coefficient and the set of statistically significant consecutive association constants which give the optimum least squares fit of the data. Application of this method indicates that the association extends at least to the tetramer.The best model for this process is that of a stepwise association for which the stepwise constants are of similar magnitudes and for which the upper limit of association is undefined. This is essentially equivalent to the indefinite model.Frontal analysis using gel filtration gives results compatible with the above model.
Purification and Properties of an Extracellular Proteinase of Psychrophilic Escherichia freundii
Authors: Nakajima Motoo , Mizusawa Kiyoshi , Yoshida Fumihiko 
Published: March 2005

A proteinase was purified from the culture filtrate of psychrophilic Escherichia freundii, by a combination of procedures, such as precipitation with ammonium sulfate, batchwise treatment with CM‐cellulose, chromatography on DEAE‐Sephadex and gel filtration on Sephadex G‐100. The final enzyme preparation was homogeneous with respect to sedimentation in the ultracentrifuge, acrylamide‐gel electrophoresis and immunodiffusion. The enzyme was an alkaline proteinase with the optimum pH at 10.0. The optimum temperature was 25°C at pH 10.0. The proteinase of psychrophilic E. freundii was fairly thermolabile. The purified enzyme was inhibited by metal‐chelating agents but not by diisopropylphosphofluoridate and p‐chloromercuribenzoate.The molecular weight of the proteinase was estimated to be 45800 by sedimentation‐equilibrium studies, 45000 by gel‐filtration method and 51000 by polyacrylamide‐gel electrophoresis in the presence of sodium dodecylsulfate. The calculation of minimum molecular weight on the basis of zinc content yielded a value near 45400 which indicated the presence of 1 g‐atom zinc per mol enzyme. The proteinase had a sedimentation coefficient of 4.12 S, a diffusion coefficient of 77.4 μm2/s, a partial specific volume of 0.718 ml/g, an isoelectric point of pH 5.2, A1%1cm at 280 nm of 12.45, and an intrinsic viscosity of 0.048 dl/g. The amino‐acid composition indicated 429 residues per molecule and the absence of sulfhydryl groups and disulfide bridges. The NH2‐ terminal amino acid of the enzyme was shown to be glycine.The extracellular Escherichia proteinase has been found to be a zinc‐containing alkaline metallo‐proteinase.
Isolation and Properties of a Staphylococcal Protease, Preferentially Cleaving Glutamoyl‐Peptide Bonds
Authors: Rydén Ann‐Christine , Phllipson Lennart , Rydén Lars 
Published: March 2005

An extracellular protease of a mutant of strain 8325N of Staphylococcus aureus, was isolated by a three‐step procedure involving stepwise chromatography on DEAE‐cellulose, gradient chromatography on hydroxyapatite and gel filtration on Sephadex G‐150. The enzyme was purified 250 times to homogeneity with a recovery of 20–30%.The protease has a molecular weight of about 29000 and consists of a single polypeptide chain as shown by gel filtration of native enzyme on calibrated columns of Sephadex G‐150 as well as gel filtration of denaturated enzyme on Sepharose 6‐B in 6 M guanidine‐HCl and dodecylsulfate‐polyacrylamide gel electrophoresis. Peptide maps also suggest a molecular weight around 30000. It is an acidic protein and is rich in aspartic acid and asparagine, but no cysteine or cystine can be detected. The N‐terminal sequence is Val‐Ile‐Leu‐Pro‐Asn‐Asn‐Asp. The enzyme contains no carbohydrate.The protease preferentially hydrolyzes peptide bonds on the carboxyl‐terminal side of glutamic acid. In a few cases aspartoyl bonds were also cleaved. It has no activity towards dipeptides with N‐terminal glutamic acid. The activity was not affected by divalent cations or EDTA, but was completely abolished after incubation with diisopropylfluorophosphate suggesting that the enzyme is a serine protease.The purified enzyme is stable between pH 3 and 10. It has maximum activity between pH 7.5 and 7.8 with casein as substrate and between pH 4.5 and 5.0 with hemoglobin as substrate.