Preparation of fully active ficin from Ficus glabrata by covalent chromatography and characterization of its active centre by using 2,2''-depyridyl disulphide as a reactivity probe.

1. Fully active ficin (EC 3.4.22.3) containing 1 mol of thiol with high reactivity towards 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) at pH4.5 per mol of protein was prepared from the dried latex of Ficus glabrata by covalent chromatography on a Sepharose-glutathione-2-pyridyl disulphide gel. 2. Ficin thus prepared is a mixture of ficins I-IV and ficin G, in which ficins II and III predominate. The various ficins exhibit similar reactivity characteristics towards 2-Py-S-S-2-Py. 3. Use of 2-Py-S-S-2-Py as a reactivity probe demonstrates (a) that in ficin, as in papain (EC 3.4.22.2), the active-centre thiol and imidazole groups interact to provide a nucleophilic state at pH values of approx. 6 additional to the uncomplicated thiolate ion that predominates at pH values over 9, and (b) a structural difference between ficin and papain that leads to a much higher rate of reaction of 2-Py-S-S-2-Py with ficin than with papain at pH values 3-4. This difference is suggested to include a lack in ficin of a carboxyl group conformationally equivalent to that of aspartic acid-158 in papain. 4. The high electrophilicity of the 2-Py-S-S-2PyH+ monocation allows directly the detection of the exposure of the buried thiol group of ficin at pH values below 4.     

Direct evidence for an acylated thiol as an intermediate in papain- and ficin-catalysed hydrolyses

1. Methyl thionohippurate was prepared and shown to be a specific substrate for both papain and ficin. 2. The ultraviolet-absorption properties of the acyl-enzyme intermediate for both papain and ficin with methyl thionohippurate was that expected of an acyl-thiol. The possibility that other functional groups present in papain or ficin might be the site of acylation has been excluded. 3. The change in extinction at the absorption maximum with time was as expected for the acyl-enzyme on the basis of the known Michaelis–Menten parameters for methyl thionohippurate. 4. The variation of extinction with initial substrate concentration for both papain and ficin was that expected from the Michaelis–Menten parameters. 5. The extinction of the absorption maximum of the thionohippuryl-enzyme intermediate was suppressed by the addition of methyl hippurate to the extent predicted from the Michaelis–Menten parameters. 6. The decay of the extinction for the acyl-enzyme was arrested by adjusting the pH of the solution to 2·5. 7. These experiments provide compelling evidence that the acylation by substrate of both papain and ficin takes place through a thiol residue.     

Enzyme separation techniques for the study of growth of cells from layers of bovine dental pulp.

Effects of the enzymes trypsin, papain, bromelains and ficin on bovine dental pulp tissue were studied. Minced or whole pulps were subjected to each enzyme at 17 degrees, 20 degrees and 37 degrees C for set time intervals, after which aliquots of supernatant fluid were removed for cell counts and viability tests. Pooled samples were subsequently cultured as monolayers in Eagle's MEM plus 10% calf serum. The dissociation characteristics were quite distinct for each enzyme, although quite similar between minced and whole pulp. A parallel histological study was made of the residual pulp tissue. Ficin was found to be the most suitable enzyme for future studies on the growth of isolated pulp cells from various layers of the bovine pulp, due to its even rate of cell removal, and the good initial viability and subsequent growth of the separated cells in monolayer culture. Further studies on ficin may show that it is more suitable for enzymatic separation of tissues generally than the more commonly used trypsin, a major advantage being its use in media containing Ca2+ and Mg2+.     

The preparation and properties of ficin chemically attached to carboxymethylcellulose

1. Purified ficin has been coupled to four CM-celluloses by reaction with their acid azide derivatives. Insoluble products containing 1.8-4.7mg. of ficin/100mg. of product and retaining 8.0-12.0% of the free enzyme's esterase activity have been obtained. 2. The amount of bound ficin in these preparations is dependent on the degree of carboxymethyl substitution of the CM-cellulose to which the ficin is attached. 3. A shift of the alkaline limb of the pH-activity curve of ficin when chemically attached to CM-cellulose has been shown. 4. Only a small loss has been observed in the enzymic activity of these products when stored at 2 degrees for 4 months. They are more resistant than free enzyme to heat denaturation. 5. Columns of CM-cellulose-ficin have been packed. The degree of hydrolysis of perfused substrate has been measured for different flow rates through the column. 6. The properties of these derivatives have been discussed.     

Selective and reversible thiol-pegylation, an effective approach for purification and characterization of five fully active ficin (iso)forms from Ficus carica latex

The latex of Ficus carica constitutes an important source of many proteolytic components known under the general term of ficin (EC 3.4.22.3) which belongs to the cysteine proteases of the papain family. So far, no data on the purification and characterization of individual forms of these proteases are available. An effective strategy was used to fractionate and purify to homogeneity five ficin forms, designated A, B, C, D1 and D2 according to their sequence of elution from a cation-exchange chromatographic support. Following rapid fractionation on a SP-Sepharose Fast Flow column, the different ficin forms were chemically modified by a specific and reversible monomethoxypolyethylene glycol (mPEG) reagent. In comparison with their un-derivatized counterparts, the mPEG-protein derivatives behaved differently on the ion-exchanger, allowing us for the first time to obtain five highly purified ficin molecular species titrating 1mol of thiol group per mole of enzyme. The purified ficins were characterized by de novo peptide sequencing and peptide mass fingerprinting analyzes, using mass spectrometry. Circular dichroism measurements indicated that all five ficins were highly structured, both in term of secondary and tertiary structure. Furthermore, analysis of far-UV CD spectra allowed calculation of their secondary structural content. Both these data and the molecular masses determined by MS reinforce the view that the enzymes belong to the family of papain-like proteases. The five ficin forms also displayed different specific amidase activities against small synthetic substrates like dl-BAPNA and Boc-Ala-Ala-Gly-pNA, suggesting some differences in their active site organization. Enzymatic activity of the five ficin forms was completely inhibited by specific cysteine and cysteine/serine proteases inhibitors but was unaffected by specific serine, aspartic and metallo proteases inhibitors.     

Purification of ficin by affinity chromatography

The sulfhydryl proteinase ficin (EC 3.4.4.12) was purified by chromatography on an agarose-mercurial column. Two separate protein fractions were eluted, ficin and mercurificin, both exhibiting enzymatic activity upon activation by excess thiol.     

Surface Plasmon Resonance Imaging biosensor for cystatin determination based on the application of bromelain, ficin and chymopapain

A Surface Plasmon Resonance Imaging (SPRI) sensor based on bromelain or chymopapain or ficin has been developed for specific cystatin determination. Cystatin was captured from a solution by immobilized bromelain or chymopapain or ficin due to the formation of an enzyme-inhibitor complex on the biosensor surface. The influence of bromelain, chymopapain or ficin concentration, as well as the pH of the interaction on the SPRI signal, was investigated and optimized. Sensor dynamic response range is between 0-0.6 μg/ml and the detection limit is equal to 0.1 μg/ml. In order to demonstrate the sensor potential, cystatin was determined in blood plasma, urine and saliva, showing good agreement with the data reported in the literature.     

Acidic cysteine proteinase inhibitor in seminal plasma

A cysteine proteinase inhibitor with acidic isoelectric point (pI = 4.7-5.0) was found in human seminal plasma. Its apparent molecular mass is 16 kDa. It inhibits cysteine proteinases like ficin, cathepsin H, cathepsin B and papain. The inhibitory activity of seminal plasma against ficin is almost the same as that of human serum.     

Evidence for a two-state transition in papain that may have no close analogue in ficin. Differences in the disposition of cationic sites and hydrophobic binding areas in the active centres of papain and ficin.

The kinetics of the reactions of the active-centre thiol groups of papain (EC 3.4.22.2) and ficin (EC 3.4.22.3) with the two-protonic-state reactivity probes 2,2'-dipyridyl disulphide, n-propyl 2-pyridyl disulphide and 4-(N-aminoethyl 2'-pyridyl disulphide)- 7-nitrobenzo-2-oxa-1,3-diazole (compound I) were studied over a wide range of pH. Differences between the reactivities of ficin and papain towards the cationic forms of the alkyl 2-pyridyl disulphide probes suggest that ficin contains a cationic site without exact analogue in papain, and the striking difference in the shapes of the pH-rate profiles for the reactions of the two enzymes with compound (1) suggests differences in the mobilities or dispositions of the active-centre histidine imidazole groups with respect to relevant hydrophobic binding areas. The evidence from reactivity-probe studies that the papain catalytic mechanism involves substantial repositioning of the active-centre imidazole group during the catalytic act does not apply also to ficin. If ficin contains an aspartic acid residue analogous to aspartic acid-158 in papain, the pKa of its carboxy group is probably significantly lower than the pKa of the analogous group in papain.     

ACTION OF PAPAIN AND FICIN ON TADPOLES AND ARBACIA EGGS

Living tadpoles and Arbacia eggs are not digested by ficin or papain although the dead organisms are. Arbacia eggs develop in papain solutions but the cells become separated. Development is normal in ficin and trypsin solutions.     

Quantitative structure-activity relationships of cysteine hydrolases. Ficin hydrolysis of X-phenyl-N-methanesulfonyl glycinates

The hydrolysis of ficin of 33 X-Phenyl-N-methanesulfonyl glycinates has been studied. The resulting Km-values have been used to derive a quantitative structure-activity relationship (QSAR). The QSAR for ficin is compared with QSAR for other cysteine hydrolases. The comparisons show that although there are specific differences, overall the reaction mechanisms are very similar.     

交联葡聚糖结合无花果蛋白酶的制备与性质

本文报道了将Sephadgx G-200与对β-硫酸酯乙砜基苯胺(SESA)首先醚化制备对氨基苯砜乙基交联葡聚糖(ABSE-Sephadex G-200),然后经重氮化固定无花果蛋白酶。固定化酶的活力回收达69％。BANA对该酶固定化过程中的活性变化有保护作用。天然酶与固定化酶都具有良好的耐热性,在69°～70℃,80min固定化酶较天然酶稳定。 用苯甲酰-DL-精氨酰-β-荼胺(BANA)为底物,在半胱氨酸存在下,测定了两种形式酶的动力学性质。在pH7.7的磷酸盐缓冲系统中,37℃,天然无花果蛋白酶的K_m=0.32mol/L;在间歇振摇下固定化酶的表观K′m=1.02mmol/L。最适pH无明显改变,均为pH7.7。     

4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole as a reactivity probe for the investigation of the thiol proteinases. evidence that ficin and bromelain may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain.

1. 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride) was used as a reactivity probe to characterize the active centres of papin (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). 2. In the pH range 0-8 Nbd chloride probably exists mainly as a monocation, possibly with the proton located on N-1 of the oxadiazole ring. 3. Spectroscopic evidence is presented for the intermediacy of Meisenheimer-type adducts in the reaction of Nbd chloride with nucleophiles. 4. The pH-dependence of the second-order rate constants (k) of the reactions of the three enzymes with Nbd chloride was determined at 25 degrees C, I = 0.1 mol/litre in 6.7% (v/v) ethanol in the pH range 2.5-5, where, at least for papain and ficin, the reactions occur specifically with their active-centre thiol groups. The pH-k profile for the papain reaction is bell-shaped (pKaI = 3.24, pKaII = 3.44 and k = 86M(-1)-s(-1), whereas that for ficin is sigmoidal (pKa = 3.6, k = 0.36M(-1)-s(-1), the rate increasing with increasing pH. The profile for the bromelain reaction appears to resemble that for the ficin reaction, but is complicated by amino-group labelling. 5. The bell-shaped profile of the papain reaction is considered to arise from the reaction of the thiolate ion of cysteine-25, maintained in acidic media by interaction with the side chain of histidine-159, with the Nbd chloride monocation hydrogen-bonded at its nitro group to the un-ionized form of the carboxyl group of aspartic acid-158. The lack of acid catalysis in the corresponding reactions of ficin and probably of bromelain suggests that these enzymes may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain. The possible consequences of this for the catalytic sites of these enzymes is discussed.     

Enzyme separation techniques for the study of growth of cells from layers of bovine dental pulp

Summary Effects of the enzymes trypsin, papain, bromelains and ficin in bovine dental pulp tissue were studied. Minced or whole pulps were subjected to each enzyme at 17°, 20° and 37°C for set time intervals, after which aliquots of supernatant fluid were removed for cell counts and viability tests. Pooled samples were subsequently cultured as monolayers in Eagle’s MEM plus 10% calf serum. The dissociation characteristics were quite distinct for each enzyme, although quite similar between minced and whole pulp. A parallel histological study was made of the residual pulp tissue. Ficin was found to be the most suitable enzyme for future studies on the growth of isolated pulp cells from various layers of the bovine pulp, due to its even rate of cell removal, and the good initial viability and subsequent growth of the separated cells in monolayer culture. Further studies on ficin may show that it is more suitable for enzymatic separation of tissues generally than the more commonly used trypsin, a major advantage being its use in media containing Ca²⁺ and Mg²⁺. Supported in part by NIH Grant No. DE 02908 and by United Health Foundation Summer Fellowships to J.T.F. and J.F.C. Supported in part by N.I.H. Grant 5 R01-DE 02908 to W.A.M. and by United Health Foundation Summer Fellowships to J.T.F. and J.F.C.     

L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide--a chromogenic substrate for thiol proteinase assay

L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA)--a convenient chromogenic substrate for assay of thiol proteinases papain, ficin, and bromelain--was prepared by enzymatic synthesis with chymotrypsin as a catalyst. The thiol proteinases hydrolyze PFLNA with the liberation of p-nitroaniline, estimated spectrophotometrically by its absorbance at 410 nm. The phenylalanine residue in the P2 position of PFLNA meets the specificity demands of thiol proteinases. The following values of Km were found for PFLNA hydrolysis: by papain, 0.34 mM; by ficin, 0.43 mM; by bromelain, 0.30 mM. This substrate was successfully applied to monitor thiol proteinase affinity chromatography on bacitracin-Sepharose, which resulted in a 2- to 4-fold purification from commercial preparations.     

Thermal Inactivation of Cysteine Proteases: The Key Stages

Biophysics - Thermal inactivation of ficin, bromelain, and papain was studied in solution and upon their immobilization on matrices of medium- (200 kDa) and high-molecular-weight (350 kDa)...     

The Effect of Proteolytic Enzymes on E. coli Phages and on Native Proteins

Lambda coli phage is not inactivated by chymotrypsin, trypsin, or ficin. T₂ phage is slowly inactivated by high concentrations of (α-, β-, γ-, or Δ-chymotrypsin, but not by trypsin or ficin. P₁ phage is slowly inactivated by α-, β-, or γ-chymotrypsin, or ficin, more rapidly by Δ-chymotrypsin, and much more rapidly by trypsin. Crystalline egg albumin, crystalline serum albumin, E. coli nucleoprotein, and yeast nucleoprotein are hydrolyzed slowly by α-chymotrypsin. Yeast nucleoprotein, like P₁ phage, is hydrolyzed more rapidly by Δ-chymotrypsin than by α-chymotrypsin, but not by trypsin or ficin. Neither phages nor native proteins were attacked by papain, carboxypeptidase, deoxyribonuclease, or ribonuclease.     

Multiple molecular forms of ficin-evidence against autolysis as explanation

While the total amount of proteolytic enzyme activity in the latex from Ficus carica varieties Kadota and Black Mission showed seasonal variation and was different between the 2 crops of figs in the latex collected at the same time and from the same tree, there is no difference in the number of multiple molecular forms of ficin present as demonstrated by chromatography on carboxymethyl-cellulose. The number of multiple forms of ficin is also the same, with 1 exception, among samples of latex which have been collected with no protection against proteolysis, samples which have been collected directly into sodium p-chloromercuribenzoate to inhibit all proteolysis and samples which have been held at an elevated temperature so as to encourage proteolysis. Evidence is presented to support the proposal that component C of Ficus carica variety Kadota is not present in the original latex but that it is produced from component D during the purification procedure. The data support the hypothesis that, with the possible exception of 1 component, the multiple molecular forms of ficin are not the result of artifacts produced by autolysis during collection, storage and purification of the sample.     

Study of the thermal denaturation of ribonuclease A by differential thermal analysis and susceptibility to proteolysis

1. The thermally induced change in conformation of ribonuclease A in solution was investigated by differential thermal analysis and the susceptibility of the enzyme to proteolytic digestion by ficin. 2. A transition with a mid-point of 60.5°C at pH4.2 was observed directly by differential thermal analysis and shown to be a property of the native structure. 3. At pH4.2 ribonuclease A is susceptible to ficin digestion at 60°C but not at 18°C. 4. Chromatographic analysis of the digestion products reveals that transient active intermediates are produced during the digestion. 5. Three of these intermediates were purified and partially characterized. 6. The nature of those sections of the ribonuclease molecule that are involved in the thermal transition is discussed.     

Affinity chromatography of a cysteine proteinase inhibitor from chicken egg protein

A method of affinity chromatography of the inhibitor of cysteine proteinases from chick egg protein using immobilized ficin has been developed. This method yields a highly active inhibitor in an essentially homogeneous state. The molecular weight of the inhibitor is 14,000. The inhibitor suppresses the activity of ficin and papain but produces no effect on the proteolytic activity of trypsin, chymotrypsin, Asp. oryzae serine proteinase or subtilisine. Isoelectric focusing of the inhibitor has revealed the major band with pI 4.35.