Phenothiazine-N-carbonyl chloride, a specific inactivator of chymotrypsin

Phenothiazine-N-carbonyl chloride inactivated chymotrypsin and trypsin by means of a 1:1 stoicheiometric reaction. Its reaction with chymotrypsin was 29 times as fast as that with trypsin and was inhibited by indole. The reaction of phenothiazine-N-carbonyl chloride with chymotrypsin resembled an enzyme-substrate reaction in which the deacylation step is rate-limiting. Slow deacylation occurred, resulting in complete regeneration of active enzyme in 15h. The pH-rate profile of the inactivation process had a maximum at pH7.8. These data and other evidence indicate that the reaction of phenothiazine-N-carbonyl chloride with chymotrypsin exhibits ;kinetic specificity'. Therefore any hypothesis that attempts to describe the topography of the active site of chymotrypsin should take into account the reactivity of phenothiazine-N-carbonyl chloride. The above findings, as well as recent reports of others, are examined within the context of a hypothesis given in an earlier paper (Erlanger, 1967).     

Solid surface-catalysed inactivation of bovine alpha-chymotrypsin in dilute solution

Although loss of chymotrypsin activity in dilute solution deviated from first order kinetics at low enzyme concentration, it displayed first order kinetics at concentrations more than 4 nM. First order rate constants varied with the ratio of surface area to volume, with the kind of vessel containing the enzyme, and with the particular test material (DS, polybrene, lecithin or BSA) coating the vessel. The reaction was saturable at lower chymotrypsin concentrations in glass than in polypropylene tubes, while less enzyme was lost at high concentrations. All these facts showed that loss of enzyme activity is incompletely, but markedly, due to a solid surface-catalysed reaction. Intrinsic fluorescence of native chymotrypsin at pH values 8 and 3, and of active site-blocked enzyme, decreased with time at 37 degrees C. No extensive autolysis of chymotrypsin was observed during the time-dependent loss of enzyme activity. Therefore, the apparent loss of chymotrypsin activity in dilute solution was mainly due to an irreversible conformational change of the molecules, as associated with the solid-surface-catalysed reaction.     

Leucine Aminopeptidase from Swine Kidney: Purification,Molecular Weight,Subunit, and Amino Acid Composition

A homogeneous leucine aminopeptidase was obtained from mixed breed swine kidneys by means of chromatography on a special column. After coupling an inhibitor, N-sulfanilyl N′-butylcarbamide, to Sepharose 6B, the derivative did not absorb the enzyme, but absorbed a non-enzymatically active protein. The enzyme showed a single band on disc-gel electrophoresis. The molecular weight of the enzyme has 320, 000 daltons. In 6 M guanidine solution containing 0.5% 2-mercaptoethanol at pH 8, the enzyme exhibited a molecular weight of 53, 000 on equilibrium centrifugation. A similar value, 54, 000, for the subunit of the enzyme was found on SDS-gel electrophoresis. The amino acid composition of the enzyme is also reported.     

Interaction of trypsin-like protease from Streptomyces griseus with an immobilized inhibitor from kidney bean.

An immobilized double-headed inhibitor from Phaseolus vulgaris L. selectively binds the trypsin-like enzyme produced by Streptomyces griseus. Binding takes place at pH 8.0, and at pH 2.0 the protease can be quantitatively released from the complex. Purified by affinity chromatography, the trypsin-like enzyme is homogeneous according to polyacrylamide gel electrophoresis and ultracentrifugation data. Physico-chemical and enzymic properties of the enzyme are identical to those exhibited by the enzyme purified by ion-exchange chromatography. Chymoelastases from Str. griseus as well as the subtilisin-like enzyme do not interact with an immobilized inhibitor. In solution, the inhibitor from P. vulgaris gives a stable ternary complex with bovine trypsin and chymotrypsin, whereas with an immobilized inhibitor the trypsin, if present, tends to displace chymotrypsin in an chymotrypsin inhibitor complex. This evidence suggests that immobilization results in considerable changes in inhibitor properties.     

Milk Clotting Enzyme from Microorganisms

The purification of the milk clotting enzyme from Mucor pusillus Lindt could be achieved by column chromatography on Amberlite IRC-50 by raising pH from 3.5 to 4.5 and about 70% of activity was recovered after this treatment. After the treatment through the column of DEAE-Sephadex A-25, the trace cellulase activity could be eliminated.

The homogeneity of the purified preparation was proved by ultracentrifugal analysis and electrophoretic patterns at various pH values.

Isoelectric point of this enzyme is considered to lie between pH 3.5 and 3.8.

The enzyme activity was inhibited by Hg⁺⁺ or Fe⁺⁺⁺.

Trypsinogenkinase activity was not contained in this enzyme.

The antiserum against the milk clotting enzyme from Mucor pusillus reacted with the purified and crude enzyme preparations in precipitin test and inhibited their enzyme activities, but did not react with other enzymes such as rennin, pepsin, acid proteases from Aspergillus saitoi and Aspergillus oryzae, or the culture filtrates of some strains of Mucor and Rhizopus.

The antigen-antibody reaction was so specific that it might be possible with this antibody to identify this enzyme and also the strain itself.

Normal sera from some mammals inhibited this enzyme activity too, but the degree was less than that with rennin.     

Human cathepsin G. Catalytic and immunological properties.

1. The specificity of cathepsin G, a neutral proteinase from human spleen, was examined by use of low-molecular-weight substrates. The enzyme was found to hydrolyse several synthetic substrates also hydrolysed by chymotrypsin, but with different kinetic constants. 2. Maximal activity against benzoyl-DL-phenylalanine 2-naphthol ester and azo-casein was in the range pH 7.5-8.0. 3. The sensitivity of cathepsin G to the action of potential inhibitors was determined, and compared with those of bovine chymotrypsin and subtilisin. Cathepsin G showed the characteristics of a serine proteinase, but was less affected by the chloromethyl ketone of tosylphenylalanine than was chymotrypsin. 4. A rabbit anti-(human cathepsin G) serum was raised, and precipitin lines formed in agarose gel were stained for activity of the enzyme. 5. Cathepsin G was shown to be immunologically identical with the chymotrypsin-like enzyme of the azurophil granules of the neutrophil granulocytes.     

Purification and characterization of cationic chymotrypsin from the pancreas of the Arabian camel (Camelus dromedarius)

This study reports on the purification and characterization of a cationic enzyme with chymotryptic activity from camel pancreas. The enzyme was purified 52-fold in a 48% yield by a three-step chromatographic procedure consisting of anion-exchange, cation-exchange and affinity chromatographies. The purified enzyme was homogeneous on gel isoelectric focusing and on SDS gel electrophoresis. Its isoelectric point was estimated to be 7.3 and its molecular mass was found to be 23,600 Da. The enzyme was identified as a cationic chymotrypsin according to its physiochemical properties, substrate specificity and susceptibility to inhibition. It was active towards esters of aromatic amino acids but much less active towards a leucine ester. In all cases, the k_cat values of the camel enzyme were less than the corresponding values of bovine chymotrypsin A. It also showed a lower level of kininase activity. Camel chymotrypsin was more susceptible than its bovine equivalent to inhibition by soybean trypsin inhibitor and aprotinin. It showed the same pH optimium as bovine chymotrypsin A for its esterolytic activity, but was more dependent on CaCl₂ for long-term stability.     

Preparation and characterization of soluble conjugates of defined molecular sizes prepared by linkage of pepsin and carboxypeptidase A to dextrans

Soluble conjugates of pepsin and carboxypeptidase A were prepared by covalent linkage of the enzymes to an amino derivative of dextran. By fractionating the dextran derivatives before and after enzyme coupling, three conjugates, with median Stokes radii between 4.0 and 11.7 nm and with a range of 25% of the median, were prepared from each enzyme. The pepsin and carboxypeptidase A conjugates contained about 35% and 3% protein, respectively. Both types had specific activities close to those of the native enzymes and were stable at -20 degrees C. The pH-activity curve was unaffected by linkage of either enzyme to dextran. The stabilities at 30 degrees C of pepsin at pH 6-7 and carboxypeptidase A at pH 3.5-9.0 were increased by linkage to dextran. No significant amount of unbound enzyme was released from either type of conjugate in skim milk. The molecular sizes, deduced from the intrinsic viscosities and the diffusion coefficients of all conjugates, were close enough to the Stokes radii to indicate that the molecules were approximately spherical. Physical measurements also indicated that the molecules were dextranlike and highly hydrated.     

Recombinant Lucilia sericata chymotrypsin in a topical hydrogel formulation degrades human wound eschar ex vivo

Larval biotherapy is a debridement tool used in wound management. The mechanism of action involves degradation of eschar by serine proteases including chymotrypsin within the alimentary fluids of first instar Lucilia sericata. With the rationale of obviating some limitations of biotherapy, including cost, complexity of use, and patient reticence, the present study describes a mobile hydrogel formulation containing freeze-dried recombinant L. sericata chymotrypsin designed for topical application. Neither freeze-drying nor formulation into the hydrogel significantly attenuated the measured activity of released enzyme compared to fresh-frozen enzyme in aqueous solution. Gel electrophoresis confirmed qualitatively that the chymotrypsin/hydrogel formulation both with and without supplementary urea at 10% (w) /(v) degraded human chronic wound eschar ex vivo. Mindful that the hallmark of intractability of chronic wounds is aberrant biochemistry, the pH activity profile for the enzyme/hydrogel formulation was compared with exudate pH in chronic wounds of mixed aetiology in a cohort of 48 hospital in-patients. Five patients' wounds were acidic, however, the remainder were predominantly alkaline and coincided with the pH optimum for the insect enzyme. Thus, a recombinant L. sericata chymotrypsin and hydrogel formulation could represent a pragmatic alternative to larval therapy for the management of chronic wounds.     

Structure and function of aspartate transcarbamoylase studied using chymotrypsin as a probe.

Aspartate transcarbamoylase from Escherichia coli is composed of six catalytic (c) and six regulatory (r) polypeptides. We have studied the structure and function of this enzyme using chymotrypsin as a probe. The protease inactivates the isolated catalytic subunit (c3) but has not effects on the native enzyme (c6r6). Under identical conditions, the c3r6 complex is inactivated at a much slower rate than c3. The presence of the substrate analogue succinate together with carbamoyl phosphate reduces substantially the rate of inactivation. Extended exposure to chymotrypsin converts the catalytic subunit into a partially active derivative with a fourfold higher Michaelis constant. This derivative is indistinguishable from the unmodified catalytic subnit in gell electrophoresis under nondenaturing conditions. However, in the presence of sodium dodecyl sulfate, the major fragment in the electropherogram is smaller than that of the intact catalytic polypeptide. The results could be explained by postulating the presence of a chymotrypsin-sensitive peptide bond at or near the active site. Since X-ray crystallographic studies have indicated that the active sites are located in a central cavity, the resistance of the native enzyme towards inactivation may be due to the inability of chymotrypsin to enter this cavity.     

Characteristics of Penicillinase Secretion by Growing Cells and Protoplasts of Bacillus licheniformis

Cultures of the inducible penicillinase-producing strain 749 of Bacillus licheniformis, induced with small amounts of benzylpenicillin, synthesized penicillinase at a high rate for a short period, after which the rate of synthesis slowly declined. During the period of active synthesis, the rate of secretion, as a fraction of the level of cell-bound penicillinase (which is originally high), gradually decreased to a constant level. Chloramphenicol, at a concentration (40 mug/ml) which completely inhibited synthesis of penicillinase, partially inhibited secretion if added during the period of active synthesis. During the phase of reduced synthesis, chloramphenicol was without effect on secretion. Penicillinase secretion, by actively growing cultures of the constitutive penicillinase-producing mutant 749/C, was inhibited by 75% immediately after addition of chloramphenicol. The secretion of part of the penicillinase released during active growth is probably dependent on synthesis of penicillinase, but part of the secreted penicillinase can be released in the absence of synthesis. Protoplasts were obtained from which periplasmic penicillinase has been removed, and these protoplasts were capable of substantial growth and penicillinase synthesis without lysis. At pH 7.5, there was no net incorporation of penicillinase into the cell membrane; the enzyme released was almost entirely of the exo form and was roughly equivalent to the amount of new enzyme formed. At pH 6.0, there was some incorporation of penicillinase into the plasma membrane, and approximately half of the extracellular penicillinase was in the exo form; the remainder perhaps represented membrane fragments. In the presence of chloramphenicol, a small amount of penicillinase was released at pH 7.5 as the exo form; at pH 6.0, practically none was released. We suggest that, with the removal from protoplasts of the periplasmic penicillinase-containing particles, a restriction on secretion has been lifted.     

The reactive sites of Kunitz bovine-trypsin inhibitor. Role of lysine-15 in the interaction with chymotrypsin.

Kunitz bovine trypsin inhibitor gave with alpha-chymotrypsin a stoichiometric complex stable at neutral pH. The complex has been characteristized by amino acid composition, molecular sieving and zone electrophoresis. Complete dissociation occurred at pH 4.0 as shown by gel filtration, alpha-Chymotrypsin was displaced from the complex by trypsin either in solution or by affinity chromatography on trypsin-Sepharos: alpha-chymotrypsin was recovered in the filtrate (yield about 100%) and the inhibitor was eluted from trypsin-Sepharose with 0.1 M HCl (yield: 83%). Lysine-15 of the inhibitor was shown to be involved in the interaction between alpha-chymotrypsin and the inhibitor. When the complex was maleylated, the maleylated chymotrypsin-bound inhibitor was displaced by affinity chromatography on trypsin-Sepharose. Teh recovered derivative was oxidized, subjected to tryptic hydrolysis and the products separated by peptide mapping and analyzed. The peptides were compared with those obtained with non-maleylated inhibitor and fully maleylated free inhibitor. In the fully maleylated inhibitor, the four lysyl residues of the molecule were blocked but in the maleylated chymotrypsin-bound inhibitor, Lys-15 was unmodified in contrast to Lys-26, Lys-41 and Lys-46; therefore Lys-15 is shielded by chymotrypsin in the complex. On the other hand, when inhibitor with a selectively reduced carboxamidomethylated Cys-14-Cys-38 dislufide bridge was allowed to react with chymotrypsin, cleavage occurred not only at Tyr-21, Tyr-35 and Phe-45 but also at Lys-15, cleavage not observed in the case of the fully oxidized inhibitor. This result shows that under particular conditions the bond Lys-15-Ala-16 can be the substrate for chymotrypsin and the side chain of Lys-15 can be inserted in the chymotrypsin specificity pocket. Apparently the contact area of inhibitor with chymotrypsin seems to be similar to that with trypsin [J. Chauvet and R. Acher (1967) J. Biol. Chem. 242, 4274-4275].     

Purification and characterization of a novel alpha-L-arabinofuranosidase from Pichia capsulata X91

An intracellular alpha-L-arabinofuranosidase from Pichia capsulata X91 was purified and characterized. The enzyme was purified to homogeneity from a cell-free extract by ammonium sulfate treatment, Concanavalin A-Sepharose, ion-exchange chromatography with DEAE Bio-Gel A agarose, arabinose-Sepharose 6B affinity chromatography, and hydroxyapatite column chromatography. The apparent molecular mass of the enzyme was estimated to be 250 kDa by native-PAGE. The enzyme molecule was suggested to be a tetramer with a subunit molecular mass of 72 kDa by SDS-PAGE. The enzyme had an isoelectric point at 5.1, and was most active at pH 6.0 and at around 50 degrees C. The alpha-L-arabinofuranosidase was active at ethanol concentrations of wine. The enzyme was inhibited by Cu2+, Hg2+, and p-chloromercuribenzoate. The enzyme hydrolyzed beet arabinan and arabinogalactan, and efficiently released monoterpenols from an aroma precursor extracted from Muscat grape juice. A considerable amount of monoterpenols was produced in the Muscat wine coupled with the enzyme addition.     

Variations of Human Milk Protein Preparations from Individual Milk Samples

The acid coagulability of casein from 54 individual human milk samples and the variation of coagulability of their casein preparations by rennin were examined. The casein and whey protein preparations from individual human milk samples were also compared by polyacrylamide gel electrophoresis (PAE). Casein coagulated distinctly from 22 human milk samples when the pH was adjusted to 4.6 with acid, but it did not from other 32 samples. Twenty-two samples of casein preparations coagulated distinctly by rennin in the presence of calcium ions but 19 samples just became turbid. When the classification of human casein based on PAE pattern of major six bands was applied in our preparations, type A appeared most often and type C did least. Any regular relationship was not found between variation of the PAE pattern of casein preparations from individual human milk samples and that of acid coagulability or rennin coagulability.     

High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. I. The hydrogen bonded protons of the "charge relay" system

High resolution proton nuclear magnetic resonance has been used to observe protons at the active site of chymotrypsin A_δ and at the same region of chymotrypsinogen A. A single resonance with the intensity of one proton is located in the low field region of the nuclear magnetic resonance spectrum. This resonance is observed in H₂O solutions but not in ²H₂O. On going from low to high pH the resonance titrates upfield 3 parts per million in both proteins and has a pK of 7.5. The titration can be prevented by alkylating His57 with either of two active site directed chloromethyl ketones. Using these data the proton resonance has been assigned to a proton in a hydrogen bond between His57 and Asp102. Further confirmation of this assignment lies in the observation of a similar resonance in this same low field region of the nuclear magnetic resonance spectrum of trypsin, trypsinogen, subtilisin BPN′ and α-lytic protease all of which have the Asp-His-Ser triad at their active sites.This proton resonance in chymotrypsin A_δ was used as a probe to monitor the charge state of the active site upon formation of a stable acyl-enzyme analogue N²(N-acetylalanyl)-N¹benzoylcarbazoyl-chymotrypsin A_δ. In this derivative the His-Asp proton resonance titrates from the same low pH end point as in the native enzyme, ?18 parts per million, to a new high pH end point of ?14.4 parts per million (versus ?15.0 parts per million in the native enzyme). The difference of 0.6 parts per million in the high pH end points between the native and acyl enzyme is interpreted as supporting the suggestion that a hydrogen bond exists between Ser195 and His57 in the native enzyme and zymogen.We conclude from these studies that the charge relay system from Asp102 across His57 to Ser195 is intact in chymotrypsin A_δ and chymotrypsinogen A, and that, in the native enzyme, it slightly polarizes Ser195.     

Affinity chromatography of enzyme cofactors: the separation of NAD on immobilised dehydrogenase colums.

1. Alcohol dehydrogenase (EC 1.1.1.1.) has been immobilised to aminoethyl-cellulose by glutaraldehyde, to DEAE-cellulose by an s-triazine derivative and to agarose using CNBr. Lactate dehydrogenase has been immobilised to the latter two supports. 2. Their use for affinity chromatography of NAD was compared and alcohol dehydrogenase immobilised to CNBr-activated agarose chosen for detailed study due to the efficient coupling of applied enzyme and the specific nature of binding. 3. The efficiency of coupling of alcohol dehydrogenase dropped from 94.5 to 72.2% when the applied load was increased from 18 to 54 mg/g activated agarose. Activity relative to free enzyme fell from 21 to 11%. The binding of NAD was maximal between pH 5.5 and 6. With the lowest loading of enzyme, NAD binding fell from 450 to 320 mug/g support when the linear flow rate was increased from 0.84 to 3.95 cm/min. 4. NAD was completely separated from a mixture with ATP, ADP and AMP. Separation from NMN and hydrolysed RNA and DNA was evidently possible. Immobilised alcohol dehydrogenase used for 34 binding experiments over a period of weeks maintained 60% of its original enzyme activity. 5. The method was applied to yeast NAD following mechanical disruption of yeast, clarification and either ultrafiltration or hollow-fibre dialysis to permit separate purification of macromolecules and nucleotides.     

Characterization of the interaction between chymotrypsin and heparin.

Heparin forms a complex with chymotrypsin which is active towards glutaryl-L-phenylalanine-p-nitroanilide (GPANA) and glutaryl-L-phenylalanine-beta-naphthylamide (GPNA) at pH 7.6. The activity of chymotrypsin towards GPANA at pH 7.6 is enhanced in the presence of heparin. Heparin does not bind at the active site of the enzyme since proflavin is not displaced from the active site of chymotrypsin upon complex formation. The heparin-chymotrypsin complex migrates under basic polyacrylamide disc gel electrophoresis conditions to a position intermediate between heparin and free chymotrypsin. The complex is dissociable under acidic polyacrylamide gel electrophoresis conditions. It is estimated that one to three molecules of heparin can bind to each chymotrypsin molecule on the basis of electrophoretic and enzymic activity data.     

A new method for determining the absolute molarity of solutions of trypsin and chymotrypsin by using p-nitrophenyl N2-acetyl-N1-benzylcarbazate

1. p-Nitrophenyl N(2)-acetyl-N(1)-benzylcarbazate (NPABC) was synthesized and shown to acylate alpha-chymotrypsin stoicheiometrically; reaction at 25 degrees occurs almost instantaneously at pH7.04 and within 2min. at pH5.04 and there is no observable turnover during 10min. 2. The absolute molarity of solutions of alpha-chymotrypsin can be determined by spectrophotometric measurement of the p-nitrophenol liberated during the acylation step; the results obtained at pH5.04 and pH7.04 agree with one another and with those determined by the method of Erlanger & Edel (1964). 3. Trypsin reacts stoicheiometrically, but more slowly than alpha-chymotrypsin, with NPABC, and it, like chymotrypsin, can be spectrophotometrically titrated at pH7.04. At pH5.04, however, reaction between trypsin and NPABC is sufficiently slow for the reagent to be nearly specific for alpha-chymotrypsin. Specificity for one or other enzyme can be ensured by using soya-bean trypsin inhibitor or the chymotrypsin inhibitor l-1-chloro-3-toluene-p-sulphonamido-4-phenylbutan-2-one. Bovine thrombin does not react with NPABC. 4. Evidence is presented that indicates that acylation of alpha-chymotrypsin and trypsin by NPABC occurs at the active centres of the enzymes. 5. Evidence was obtained that indicates that one or more tryptophan residues move into a more hydrophobic environment when alpha-chymotrypsin and trypsin are acylated by NPABC.     

The structure and function of acid proteases. V. Comparative studies on the specific inhibition of acid proteases by diazoacetyl-DL-norleucine methyl ester, 1,2-epoxy-3-(p-nitrophenoxy) propane and pepstatin.

Comparative studies have been made on the effects of diazoacetyl-DL-norleucine methyl ester (DAN), 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and pepstatin on acid proteases, including those from Acrocylindrium sp., Aspergillus niger, Aspergillus saitoi, Mucor pusillus, Paecilomyces varioti, Rhizopus chinensis, and Trametes sanguinea, and also porcine pepsin [EC 3.4.23.1] and calf rennin [EC 3.4.23.4] for comparative purposes. These enzymes were rapidly inactivated at similar rates and in 1:1 stiochiometry by reaction with DAN in the presence of cupric ions. The pH profiles of inactivation of these enzymes were similar and had optima at pH 5.5 to 6. They were also inactivated at similar rates by reaction with EPNP, with concomitant incorporation of nearly 2 EPNP molecules per molecule of enzyme. The pH profiles of inactivation were again similar and maximal inactivation was observed at around pH 3 to 4. Some of the EPNP-inactivated enzymes were treated with DAN and shown still to retain reactivity toward DAN. All these enzymes were inhibited strongly by pepstatin, and the reactions of DAN and EPNP with them were also markedly inhibited by prior treatment with pepstatin. These results indicate that the active sites of these enzymes are quite similar and that they presumably have at least two essential carboxyl groups at the active site in common, one reactive with DAN in the presence of cupric ions and the other reactive with EPNP, as has already been demonstrated for porcine pepsin and calf rennin. Pepstatin appears to bind at least part of the active site of each enzyme in a simmilar manner.     

The structure and function of ribonuclease T1 XXIV. Preparation and properties of a stable water-insoluble polyacrylamide derivative of ribonuclease T1.

Ribonuclease T1 [EC 3.1.4.8] was coupled to a water-insoluble cross-linked polyacrylamide (Enzacryl AH) by the acid azide method. The immobilized enzyme exhibited about 45% and 77% of the original activity toward yeast RNA and 2', 3-cyclic GMP, respectively, as substrates. Although the specific activity was lowered by the coupling, the immobilized enzyme was found to be far more stable to heat and extremes of PH than the native enzyme. The immobilized enzyme was active toward RNA even above pH 9 (at 37 degree C) or above 60 degree C (at pH 7.5), where the native enzyme was inactive. The immobilized enzyme retained much of its activity as assayed at 37 degree C after incubation in the range of pH 1 to 10 at 37 degree C, or after heating at 100 degree C (at pH 7.5) under conditions where the native enzyme was inactivated to a considerable extent. The enzyme derivative could be repeatedly recovered and reused without much loss of activity. The active site glutamic acid-58 in the immobilized enzyme appeared to be nearly as reactive with iodoacetate as that in the native enzyme.