Papaya Latex Enzyme

The changes in the heme environment and overall structure occurring during reversible thermal inactivation and in denaturant guanidinium of Euphorbia characias latex peroxidase (ELP) were investigated in the presence and absence of calcium ions. Native active enzyme had an absorption spectrum typical of a quantum-mixed spin ferric heme protein. After 40 min at 60 • C ELP was fully inactivated showing the spectroscopic behavior of a pure hexacoordinate low-spin protein. The addition of Ca 2+ to the thermally inactivated enzyme restored its native activity and its spectroscopic features, but did not increase the stability of the protein in guanidinium. It is concluded that, in Euphorbia peroxidase, Ca 2+ ion play a key role in conferring structural stability to the heme environment and in retaining active site geometry.

Background: By and large Natural Rubber Latex (NRL) allergy is remarked in individuals after exposure to the material. Since NRL is usually found in various materials used in clinical and surgical practices, so NRL allergy can be manifested at day to day basis. The clinical manifestations of latex allergies can range from mild to severe, but it is important to diagnose these allergic reactions and tackle them in a proper way. Conclusion: Severe anaphylactic shocks have been reported because of Natural rubber latex. Chances of wrong diagnosis or mis-diagnosis are more when it comes to latex allergy there by risking the lives of patients. Therefore, precautions must always be taken while carrying out clinical and surgical procedures in order to achieve satisfactory therapeutic approaches and avoiding inflammatory dermatitis and anaphylactic shocks.

This article addresses the role of glove powder in facilitating allergic reactions to natural rubber latex (NRL) and to the chemical additives in synthetic and NRL gloves as well as its role in eliciting postsurgical complications. Various dusting powders have been used historically to prevent gloves from sticking to each other and to facilitate donning. All have manifested adverse consequences for health care professionals and patients. Manufacturing methods for powder reduction and elimination are presented. The recently developed ASTM methods for the quantitation of powder on powder-free and powdered gloves are reviewed along with the new ASTM maximum powder limits for all medical gloves. Caution must be exercised when methods of protein and powder reduction are implemented to minimize the possibility of creating other adverse consequences.

Antioxidant activities of gelatin hydrolysates from blacktip shark skin prepared using papaya latex enzyme with different degrees of hydrolysis (DHs: 10%, 20%, 30% and 40%) were evaluated. All antioxidant activity indices of hydrolysates increased with increasing DH (P < 0.05). When gelatin hydrolysate with 40%DH was determined for its pH and thermal stability, ORAC and chelating activity remained constant or slightly increased in a wide pH range (1-9) and during heating (100°C) for 240 min. It was also stable in simulated gastrointestinal tract model system. Moreover, gelatin hydrolysate at a level of 500 and 1000 ppm could inhibit lipid oxidation in both b-carotene linoleate and cooked comminuted pork model systems. Therefore, gelatin hydrolysate from blacktip shark skin (40%DH) can potentially be used as an alternative source of natural antioxidants.

Peroxidases (POXs) and Catalases (CATs) are the main antioxidant enzymes involved in scavenging H2O2 in living cells. Different POXs and CATs may be capable of exhibiting interaction with the constituents of the plant cell. Whereas the activity or gene expression of POXs and CATs has been investigated in potato plants, their interactions with other proteins in this crop have not been investigated till now. Determining Protein-Protein Interaction (PPI) networks could be important in providing crucial insights into the regulation of plant defense responses to biotic and abiotic stresses. STRING analysis revealed interaction of cationic, suberization-associated anionic, and Class III peroxidases in potato with several enzymes involved in lignin biosynthesis and phenylpropanoid pathways, which was in accordance with close phylogenetic relationship of the three potato peroxidases investigated in this study. The CAT1 enzyme in potato interacted with several enzymes involved in ROS product...

Perioperative immediate hypersensitivity reactions represent rare but not negligible complications during surgery, even in a pediatric environment. Latex is the first cause of hypersensitivity reactions during anesthesia in children. We present the case of a 10 years old asthmatic boy who was labeled as allergic to latex, after an anaphylactic shock occurring right after a general anesthesia. A proper allergy work-up allowed us to rule out such a diagnosis and to precisely advise the anesthesiologist and the surgical team on how to perform a safe intervention on this patient.

An aqueous two-phase system (ATPS) in combination with ammonium sulfate ((NH 4) SO 4) precipitation was applied to fractionate glycyl endopeptidase from the papaya latex of Red Lady and Khack Dum cultivars. ATPS containing polyethylene glycol (PEG 2000 and 6000) and salts ((NH 4) 2 SO 4 and MgSO 4) at different concentrations were used. Glycyl endopeptidase with high purity fold (PF) and yield was found in the salt-rich bottom phase of ATPS with 10% PEG 6000-10% (NH 4) 2 SO 4. When ATPS fraction from Red Lady cultivar was further precipitated with 40-60% saturation of (NH 4) 2 SO 4 , PF of 2.1-fold with 80.23% yield was obtained. Almost all offensive odorous compounds, particularly benzyl isothiocyanate, were removed from partially purified glycyl endopeptidase (PPGE). The fish gelatin hydrolysates prepared using PPGE showed higher ABTS radical scavenging activity and less odour, compared with those of crude extract (CE). Thus antioxidative gelatin hydrolysate with negligible undesirable odour could be prepared with the aid of PPGE.

Inhibition of angiotensin converting enzyme (ACE), human LDL cholesterol and DNA oxidation by hydrolysates from blacktip shark gelatin prepared using papaya latex enzyme with different degrees of hydrolysis (DH of 10, 20, 30 and 40%) were evaluated. ACE-inhibitory activity of hydrolysate increased as the DH increased (P < 0.05). Gelatin hydrolysate (10e40%DH) at a concentration of 0.5 g L À1 inhibited LDL cholesterol oxidation by 8.32e39.21%. Additionally, gelatin hydrolysate with DH of 40% effectively inhibited hydroxyl and peroxyl radical-induced DNA scission. Therefore, gelatin hydrolysate from blacktip shark skin with DH of 40% could serve as a potential source of functional food ingredient for health promotion.

Background: Natural rubber latex and chlorhexidine have previously been identified as causative substances in perioperative anaphylaxis. A pelvic examinations is generally considered noninvasive, however, this procedure is rarely associated with severe allergic reactions. We reported a rare case of dual latex and chlorhexidine allergies which caused anaphylaxis after pelvic examination in a woman with a history of latex-related fruits allergy. Case presentation: A 54-year-old woman had severe anaphylaxis after a pelvic examination due to dual latex and chlorhexidine (CHX) allergies. The gynecologist used CHX for the vaginal preparation and wore latex-containing gloves with lubricating gel during the examination. In vivo and in vitro tests revealed CHX sensitization by a positive skin prick test to chlorhexidine at a very low concentration (0.002 mg/mL), and a positive basophil activation test to CHX. Latex allergy was confirmed by a positive specific IgE to latex and a positive glove-use test at 20 min. An analysis of specific IgE to latex component revealed positive results for Hev b 1, 5, 6.02, and 11. As she also had a past history of fruit allergy, prick-to-prick testing with latex-related fruits was performed. The results were positive for avocado, banana, jackfruit, kiwi, and longan. Conclusions: Concomitant mucosal exposure of both natural rubber latex and CHX in highly sensitized patients during pelvic examinations can lead to severe anaphylaxis. Pre-procedural screening for an allergy to latex or CHX, or to any other allergen, should be performed in patients where there is suspicion of a specific allergy due to a previous allergic reaction. Increased awareness of these two allergens in all healthcare settings may improve patient safety.

Composition, functional properties, and in vitro antioxidant activities of gelatin hydrolysates prepared from cuttlefish skin were investigated. Cuttlefish skin gelatin hydrolysates (CSGHs) were obtained by treatment with crude enzyme preparations from Bacillus licheniformis NH1, Bacillus mojavensis A21, Bacillus subtilis A26, and commercial alcalase. All CSGHs had high protein contents, 74.3-78.3%, and showed excellent solubility (over 90%). CSGH obtained by alcalase demonstrated high antioxidant activities monitored by β-carotene bleaching, DPPH radical scavenging, lipid peroxidation inhibition, and reducing power activity. Its antioxidant activity remained stable or increased in a wide range of pH (1-9), during heating treatment (100°C for 240 min) and after gastrointestinal digestion simulation. In addition, alcalase-CSGH was incorporated into turkey meat sausage to determine its effect on lipid oxidation during 35 days of storage period. At 0.5 mg/g, alcalase-CSGH delayed lipid...

Hypersensitivity reactions to natural rubber latex have increased recently, especially among people with high exposure to latex allergens. Hypersensitivity reactions to latex are related to many conditions like occupational asthma. Our study was performed to determine the prevalence of hypersensitivity to natural rubber latex and potential food cross reactions in operation room personnel in Shiraz hospitals. In this cross-sectional, descriptive study, 580 operation room personnel filled out our questionnaire which included data about their personal history, symptoms of latex hypersensitivity, and other related allergies such as food hypersensitivity. An informed consent was obtained and skin prick tests were performed for natural rubber latex and potential food cross reactions (kiwi, banana, and potato). The obtained data were analyzed by SPSS and Chi-square test.Results: 104 (17.9%) of the operating room personnel showed positive latex skin tests. We revealed a significant correlat...

eontent of various natural latex articles. Allergy 1997: 52: 661-664. © Munksgaard 1997. potential setisitizers. In the present work, we quantified the releasable proteiti atul allergeti eontents in 37 brands of lalex gloves and 26 other latex produets. Our results deniotistrate the presetiee of widely varied proteiti and allergen contents iti various latex artieles and the laek of a eorrelation between the protein and allergen values. These findings may assist hospital tiianagement and niedieal staff to take el'feetive preventive measures.

This paper deals with the purification of four proteins from Euphorbia characias latex, a copper amine oxidase, a nucleotide pyrophosphatase/phosphodiesterase, a peroxidase, and a purple acid phosphatase. These proteins, very different in molecular weight, in primary structure, and in the catalyzed reaction, are purified using identical preliminary steps of purification and by chromatographic methods. In particular, the DEAE-cellulose chromatography is used as a useful purification step for all the four enzymes. The purification methods here reported allow to obtain a high purification of all the four proteins with a good yield. This paper will give some thorough suggestions for researchers busy in separation of macromolecules from different sources.

The changes in the heme environment and overall structure occurring during reversible thermal inactivation and in denaturant guanidinium of Euphorbia characias latex peroxidase (ELP) were investigated in the presence and absence of calcium ions. Native active enzyme had an absorption spectrum typical of a quantum-mixed spin ferric heme protein. After 40 min at 60 • C ELP was fully inactivated showing the spectroscopic behavior of a pure hexacoordinate low-spin protein. The addition of Ca 2+ to the thermally inactivated enzyme restored its native activity and its spectroscopic features, but did not increase the stability of the protein in guanidinium. It is concluded that, in Euphorbia peroxidase, Ca 2+ ion play a key role in conferring structural stability to the heme environment and in retaining active site geometry. (R. Medda). found in the cytosol, vacuole, apoplast or cell wall, and are involved in the regulation of cell growth and differentiation, cell wall lignification, metabolism of hormones and alkaloids, wound healing and defense against pathogen infection . Typically, class III peroxidases may exist under an extremely high number of isoforms within the same species, potentially implicated in different functions . For example, the peroxidase isoenzyme family of Arabidopsis thaliana comprises over 70 full-length genes, most of which predicted to encode for stable enzymes .

Abbreviations: ALDH) aldehyde dehydrogenases; CaM) calmodulin; EAq) Euphorbia antiquitin; ECat) Euphorbia cata lase; ELAO) Euphorbia latex amine oxidase; ELP) Euphorbia latex peroxidase; nt) nucleotide; RACE) rapid amplification cDNA ends; RT PCR) reverse transcription polymerase chain reaction.

Composition, functional properties, and in vitro antioxidant activities of gelatin hydrolysates prepared from cuttlefish skin were investigated. Cuttlefish skin gelatin hydrolysates (CSGHs) were obtained by treatment with crude enzyme preparations from Bacillus licheniformis NH1, Bacillus mojavensis A21, Bacillus subtilis A26, and commercial alcalase. All CSGHs had high protein contents, 74.3-78.3%, and showed excellent solubility (over 90%). CSGH obtained by alcalase demonstrated high antioxidant activities monitored by β-carotene bleaching, DPPH radical scavenging, lipid peroxidation inhibition, and reducing power activity. Its antioxidant activity remained stable or increased in a wide range of pH (1-9), during heating treatment (100°C for 240 min) and after gastrointestinal digestion simulation. In addition, alcalase-CSGH was incorporated into turkey meat sausage to determine its effect on lipid oxidation during 35 days of storage period. At 0.5 mg/g, alcalase-CSGH delayed lipid...

Horseradish peroxidase A1 can refold to a native-like structure without binding calcium, originating a Ca 2+ -depleted native state as previously demonstrated. Thermal unfolding studies of horseradish peroxidase anionic 1 (HRPA1) have shown that calcium ions present during refolding lead to the appearance of a misfolded conformational state, which cannot incorporate the heme group. This calcium-induced conformational state, I Ca 2+ , is less stable than the native state and has distinct secondary and tertiary structures as probed by far-UV and visible circular dichroism and tryptophan fluorescence. The fraction of I Ca 2+ increases exponentially with increasing calcium concentration. The I Ca 2+ state is formed during refolding after calcium binding to the unfolded state, as reconstitution of HRPA1 from its apoprotein reveals that the affinity of the apoprotein to protoporphyrin IX is higher in the presence of calcium. If calcium is added after refolding only, the majority of HRPA1 molecules retain their native conformation, thus confirming the binding of calcium to the unfolded state. D

An aqueous two-phase system (ATPS) in combination with ammonium sulphate ((NH4)2SO4) precipitation
was applied to fractionate glycyl endopeptidase from the papaya latex of Red Lady and Khack
Dum cultivars. ATPS containing polyethylene glycol (PEG 2000 and 6000) and salts ((NH4)2SO4 and
MgSO4) at different concentrations were used. Glycyl endopeptidase with high purification fold (PF)
and yield was found in the salt-rich bottom phase of ATPS with 10%PEG 6000–10% (NH4)2SO4.When ATPS
fraction from Red Lady cultivar was further precipitated with 40–60% saturation of (NH4)2SO4, PF of
2.1-fold with 80.23% yield was obtained. Almost all offensive odorous compounds, particularly benzyl
isothiocyanate, were removed from partially purified glycyl endopeptidase (PPGE). The fish gelatin
hydrolysates prepared using PPGE showed higher ABTS radical scavenging activity and less odour, compared
with those of crude extract (CE). Thus antioxidative gelatin hydrolysate with negligible undesirable
odour could be prepared with the aid of PPGE.

Although severe anaphylatic reactions are rare, allergic or immediate hypersensitivity reactions to natural rubber latex (NRL) have been reported in children with increasing frequency in the last few years. Indubitably, every-day rubber has been employed in thousands and thousands of products for over a century, while recently NRL allergy has been recognized as a substantial medical problem. Children with spina bifida undergoing multiple surgical procedures, as well healthy babies, appear at high risk for NRL allergy, with life-threatening reactions reported during surgery or while playing with balloons. Skin prick testing is the preferred diagnostic method, especially in the prick by prick version. During procedures, Parents of children with latex allergy should take caution that their babies avoid contact with rubber products, and a latex-free environment. Since minimal contacts with NRL products may produce reactions, the primary treatment for latex allergy is avoidance. The cross-reactions regarding the latex-fruit syndrome have recently widened the number of possible reactions.

A new cationic peroxidase from Euphorbia tirucalli (pencil cactus) latex was purified to homogeneity using benzene fractionation, gel filtration and cation-exchange chromatography. The purified enzyme was found to be monomeric with a molecular weight of 44 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme had a broad specificity towards some phenolic substrates in the order of 2,2'azino-bis (3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) > guaiacol > o-phenylenediamine > 4-aminoantipyrene, whereas no affinity towards ascorbic acid and o-dianisidine was recorded. The enzyme had pH and temperature optima at 7.0 and 40°C, respectively. Study of kinetic parameters demonstrated that ABTS had the highest affinity towards ELP, where K m , V max and V max /K m values were 0.503 mM, 500 U/assay and 994.04 U/mM, respectively. ELP was stable from 10°C up to 60°C and lost about 70% of its activity at 70˚C. The thermal inactivation profile of ELP in absence of Ca 2+ is biphasic and characterized by a rapid decline in activity on exposure to heat, followed by a more gradual decrease in activity on continued exposure. However, the purified enzyme exhibited increased thermal stability in the presence of calcium ions. Furthermore, the activity of purified enzyme was enhanced by ~ 550% in the presence of 15 mM CaCl 2 , suggesting a pivotal role for Ca 2+ in conferring structural stability to the heme environment and in retaining the active site of Afaf S. Fahmy et all ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ ‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ ‫ــــ‬ ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ 246

Calmodulin (CaM) is a ubiquitous Ca2+ sensor found in all eukaryotes, where it participates in the regulation of diverse calcium-dependent physiological processes. In response to fluctuations of the intracellular concentration of Ca2+, CaM binds to a set of unrelated target proteins and modulates their activity. In plants, a growing number of CaM-binding proteins have been identified that apparently do not have a counterpart in animals. Some of these plant-specific Ca2+/CaM-activated proteins are known to tune the interaction between calcium and H2O2 in orchestrating plant defenses against biotic and abiotic stresses. We previously characterized a calcium-dependent peroxidase isolated from the latex of the Mediterranean shrub Euphorbia characias (ELP) [Medda et al. (2003) Biochemistry 42, 8909-8918].
Here we report the cDNA nucleotide sequence of Euphorbia latex peroxidase, showing that the derived protein has two distinct amino acid sequences recognized as CaM-binding sites. The cDNA encoding for an E. characias CaM was also found and sequenced, and its protein product was detected in the latex. Results obtained from different CaM-binding assays and the determination of steady-state parameters
showed unequivocally that ELP is a CaM-binding protein activated by the Ca2+/CaM system. To the best of our knowledge, this is the first example of a peroxidase regulated by this classic signal transduction mechanism. These findings suggest that peroxidase might be another node in the Ca2+/H2O2-mediated plant defense system, having both positive and negative effects in regulating H2O2 homeostasis

The changes in the heme environment and overall structure occurring during reversible thermal inactivation and in denaturant guanidinium of Euphorbia characias latex peroxidase (ELP) were investigated in the presence and absence of calcium ions. Native active enzyme had an absorption spectrum typical of a quantum-mixed spin ferric heme protein. After 40 min at 60°C ELP was fully inactivated showing the spectroscopic behavior of a pure hexacoordinate low-spin protein. The addition of Ca2+ to the thermally inactivated enzyme restored its native activity and its spectroscopic features, but did not increase the stability of the protein in guanidinium. It is concluded that, in Euphorbia peroxidase, Ca2+ ion play a key role in conferring structural stability to the heme environment and in retaining active site geometry

Here we report the cDNA nucleotide sequences of a calmodulin
binding catalase and an antiquitin from the latex
of the Mediterranean shrub Euphorbia characias. Present findings suggest that catalase and antiquitin might represent addi
tional nodes in the Euphorbia defense systems, and a multi
enzymatic interaction contributing to plant’s protection against biotic and abiotic stresses is proposed to occur in E. characias laticifers

Plant latex is a complex environment. Occurring in hundreds of plant species and contained in a tube system called
laticifers, latex is a milky sap with a diverse composition that includes alkaloids, terpenoid compounds, other secondary
metabolites and a number of enzymes. These substances are collectively believed to provide an important contribution to
plant defence mechanisms by repelling and killing phytopathogens, and sealing wounded areas. This review provides insights of what is currently known about the biochemistry and molecular biology of plant latex, as studied in various model systems, above all the economically important rubber tree, Hevea brasiliensis. Selecting the Mediterranean shrub Euphorbia characias as a complementary experimental model, we have recently begun to disclose the properties of several components of the enzymatic machinery present in its latex. Although the scheme of multi-enzymatic interactions taking place in the
E. characias latex depicted to date is certainly incomplete, the emerging scenario suggests that the role played by latex in plants might be significantly less passive than previously believed.

The changes in the heme environment and overall structure occurring during reversible thermal inactivation and in denaturant guanidinium of Euphorbia characias latex peroxidase (ELP) were investigated in the presence and absence of calcium ions. Native active enzyme had an absorption spectrum typical of a quantum-mixed spin ferric heme protein. After 40 min at 60 • C ELP was fully inactivated showing the spectroscopic behavior of a pure hexacoordinate low-spin protein. The addition of Ca 2+ to the thermally inactivated enzyme restored its native activity and its spectroscopic features, but did not increase the stability of the protein in guanidinium. It is concluded that, in Euphorbia peroxidase, Ca 2+ ion play a key role in conferring structural stability to the heme environment and in retaining active site geometry. (R. Medda). found in the cytosol, vacuole, apoplast or cell wall, and are involved in the regulation of cell growth and differentiation, cell wall lignification, metabolism of hormones and alkaloids, wound healing and defense against pathogen infection . Typically, class III peroxidases may exist under an extremely high number of isoforms within the same species, potentially implicated in different functions . For example, the peroxidase isoenzyme family of Arabidopsis thaliana comprises over 70 full-length genes, most of which predicted to encode for stable enzymes .

Calmodulin (CaM) is a ubiquitous Ca 2+ sensor found in all eukaryotes, where it participates in the regulation of diverse calcium-dependent physiological processes. In response to fluctuations of the intracellular concentration of Ca 2+ , CaM binds to a set of unrelated target proteins and modulates their activity. In plants, a growing number of CaM-binding proteins have been identified that apparently do not have a counterpart in animals. Some of these plant-specific Ca 2+ /CaM-activated proteins are known to tune the interaction between calcium and H 2 O 2 in orchestrating plant defenses against biotic and abiotic stresses. We previously characterized a calcium-dependent peroxidase isolated from the latex of the Mediterranean shrub Euphorbia characias (ELP) Biochemistry 42, 8909-8918].

Here we report the cDNA nucleotide sequences of a calmodulin-binding catalase and an antiquitin from the latex of the Mediterranean shrub Euphorbia characias. Present findings suggest that catalase and antiquitin might represent additional nodes in the Euphorbia defense systems, and a multi-enzymatic interaction contributing to plant’s protection against biotic and abiotic stresses is proposed to occur in E. characias laticifers.

Antioxidant activities of gelatin hydrolysates from blacktip shark skin prepared using papaya latex enzyme with different degrees of hydrolysis (DHs: 10%, 20%, 30% and 40%) were evaluated. All antioxidant activity indices of hydrolysates increased with increasing DH (P < 0.05). When gelatin hydrolysate with 40%DH was determined for its pH and thermal stability, ORAC and chelating activity remained constant or slightly increased in a wide pH range (1–9) and during heating (100 °C) for 240 min. It was also stable in simulated gastrointestinal tract model system. Moreover, gelatin hydrolysate at a level of 500 and 1000 ppm could inhibit lipid oxidation in both β-carotene linoleate and cooked comminuted pork model systems. Therefore, gelatin hydrolysate from blacktip shark skin (40%DH) can potentially be used as an alternative source of natural antioxidants.► Gelatin hydrolysate from shark skin showed high DH (40%) when prepared using papaya latex enzyme. ► DH directly affected antioxidant activity of gelatin hydrolysate, both by chain breaking and preventive mechanisms. ► Gelatin hydrolysate retarded lipid oxidation in model systems.