Plant Serine Protease Research Articles

Cutaneous Melanoma: An Overview of Physiological and Therapeutic Aspects and Biotechnological Use of Serine Protease Inhibitors.

Metastatic melanoma stands out as the most lethal form of skin cancer because of its high propensity to spread and its remarkable resistance to treatment methods. In this review article, we address the incidence of melanoma worldwide and its staging phases. We thoroughly investigate the different melanomas and their associated risk factors. In addition, we underscore the principal therapeutic goals and pharmacological methods that are currently used in the treatment of melanoma. The implementation of targeted therapies has contributed to improving the approach to patients. However, because of the emergence of resistance early in treatment, overall survival and progression-free periods continue to be limited. We provide new insights into plant serine protease inhibitor therapeutics, supporting high-throughput drug screening soon, and seeking a complementary approach to explain crucial mechanisms associated with melanoma.

Improving the antithrombogenicity of nylon monofilament for surgical suture by coating a protease from Euphorbia resinifera latex

This study developed a method to improve the antithrombogenicity of a blue nylon monofilament suture (USP 3/0) by coating a plant serine protease purified from Euphorbia resinifera latex (EuRP-61) onto the surface of nylon using a combination of dipping and ultrasonication methods. EuRP-61 was a selected protease because the enzyme had been previously reported to have antithrombotic and anticoagulant activities, high stability and biological safety. Analyses of the fibrinogenolytic and fibrinolytic activities using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and microwell plate assays revealed that the proper concentration of protease was 1 mg/ml for each coating of 20 strips of 10-mm nylon. The enzyme-coated nylon was disinfected by ethylene oxide (EO) sterilization. After EO sterilization, the enzyme-coated nylon presented fibrinogenolytic and fibrinolytic activities. Comparing the enzyme-coated and bare nylon, the enzyme-coated nylon prevented the human fibrin clot from adhering to its surface. In contrast, the fibrin clot stuck and was unable to be removed from the surface of the bare nylon. The antithrombogenicity of the protease-coated nylon evaluated in this study suggests its potential application as a surgical suture, especially for vessel anastomosis.

Identification of Phytaspase Interactors via the Proximity-Dependent Biotin-Based Identification Approach.

Proteolytic enzymes are instrumental in various aspects of plant development, including senescence. This may be due not only to their digestive activity, which enables protein utilization, but also to fulfilling regulatory functions. Indeed, for the largest family of plant serine proteases, subtilisin-like proteases (subtilases), several members of which have been implicated in leaf and plant senescence, both non-specific proteolysis and regulatory protein processing have been documented. Here, we strived to identify the protein partners of phytaspase, a plant subtilase involved in stress-induced programmed cell death that possesses a characteristic aspartate-specific hydrolytic activity and unusual localization dynamics. A proximity-dependent biotin identification approach in Nicotiana benthamiana leaves producing phytaspase fused to a non-specific biotin ligase TurboID was employed. Although the TurboID moiety appeared to be unstable in the apoplast environment, several intracellular candidate protein interactors of phytaspase were identified. These were mainly, though not exclusively, represented by soluble residents of the endoplasmic reticulum, namely endoplasmin, BiP, and calreticulin-3. For calreticultin-3, whose gene is characterized by an enhanced expression in senescing leaves, direct interaction with phytaspase was confirmed in an in vitro binding assay using purified proteins. In addition, an apparent alteration of post-translational modification of calreticultin-3 in phytaspase-overproducing plant cells was observed.

Fibrin – Fibrinogen scaffold structural modification by phytochemicals and phytoproteases contained in an aqueous extract of Ageratum conyzoides Linn.

Based on mechanisms of fibrin clot polymerization and dissolution, it is possible to modulate fibrin formation and removal. Ageratum conyzoides Linn. (Asteraceae) is an annual herb with a long history of traditional medicine. There is high variability in the secondary metabolites of this plant which include flavonoids, and these molecules belong to a class of serine proteases inhibitors. Several plant enzymes belonging to the classes of serine proteases were observed to be active on the cascade of coagulation pathways. The aim of this study was to observe if even Ageratum conyzoides Linn. aqueous leaves extract contained proteases which could structurally modify the fibrin clot formation. To prepare plant extracts, dry leaves of the plant were extracted with distilled water. Fibrin gels were prepared by mixtures containing fibrinogen and thrombin with or without extract. Fibrin networks were disrupted by a denaturation buffer. Samples were deposited in 8% polyacrylamide gel and Coomassie blue was used to reveal migration. Our extract contained phytochemicals class flavonoids which are thrombin inhibitors. But our results support the evidence that the same extract contained plant serine proteases, specifically a fibrinogenase which hydrolyzed fibrinogen but not like thrombin.Keywords: Fibrin/Fibrinogen, structural modification, Ageratum conyzoides Linn., phytoproteases.

GdTI, the first thermostable trypsin inhibitor from Geoffroea decorticans seeds. A novel natural drug with potential application in biomedicine

A novel thermostable trypsin inhibitor was obtained from Geoffroea decorticans seeds. G. decorticans trypsin inhibitor (GdTI) is a protein with molecular mass of 6743.7 Da, with a potent inhibitory activity (Ki of 2.1 nM) even at high temperatures and extreme pHs (100% after 5 h at 100 °C and 80% after 60 min at pH 2–12) constituting one of the most powerful serine protease inhibitors isolated from a plant source. GdTI displays anticoagulant activity against both extrinsic and intrinsic coagulation pathways, representing the first report of a plant serine protease inhibitor with anticoagulant activity against the extrinsic pathway. Finally, GdTI showed inhibitory activity against α-glucosidase (IC50 of 0.18 μM) evidencing the hypoglycemic effect of this inhibitor. Our results evidence the discovery of a natural molecule with unique features: i) GdTI is one of the most potent serine protease inhibitors founded to date, ii) with the most powerful thermostability reported in literature, iii) with anticoagulant effect against both coagulation pathways and hypoglycemic activity. This report suggest that GdTI could be exploited as a natural and hyperstable antidiabetic drug, in behalf of its antithrombotic and hypoglycemic activities, encouraging future studies with high impact on biomedical research and potential pharmaceutical applications.

Plant serine protease inhibitor (SPI): A potent player with bactericidal, fungicidal, nematicidal and antiviral properties

This review describes plant originated serine protease inhibitors (SPIs) that target or inhibit different families of serine proteases (SPs), protein-digesting enzymes of various pathogens. SPs play a crucial task in many biological events by catalyzing proteolysis that serves as mediators of signal initiation, transmission and termination of the cellular events leading to regulation of an organism’s life cycle. The activity of proteases has to be closely regulated by proteinase inhibitors (PIs) to avoid the damage that proteases might cause in vivo, in the host organism. Based on the selectivity and inhibitory activity PIs are categorized into four mechanistic class viz. aspartic PI (pepstatins), serine PI (serpins), cysteine PI (cystatins) and metallocarboxy PI. Among these SPI family is the largest. Many organisms including bacteria, fungus, viruses, protists, insects and vertebrates derive their nutritional requirement from various life forms by finding a suitable host. Proteases constitute 1-5% of genomes of these infectious organisms among which SPs regulate protein synthesis, turnover and physiological functions viz. fertilization, growth and development, digestion, cell signaling or migration, immune defense, wound healing and disease propagation. SPs mediate the process of pathogenesis and/or host tissue penetration for a number of diseases in the host organisms. SPs are generally found either as constitutive components in plant storage tissues like seeds and tubers or expressed in response to pest and pathogen attack besides acting as a defense system against a wide variety of pathogens. These expressed or endogenously present SPIs can attenuate SPs with cross-protection against a wide array of protease families of pathogens like bacteria, fungus, nematodes and viruses.

Plant Serine Protease Inhibitors: Biotechnology Application in Agriculture and Molecular Farming.

The serine protease inhibitors (SPIs) are widely distributed in living organisms like bacteria, fungi, plants, and humans. The main function of SPIs as protease enzymes is to regulate the proteolytic activity. In plants, most of the studies of SPIs have been focused on their physiological role. The initial studies carried out in plants showed that SPIs participate in the regulation of endogenous proteolytic processes, as the regulation of proteases in seeds. Besides, it was observed that SPIs also participate in the regulation of cell death during plant development and senescence. On the other hand, plant SPIs have an important role in plant defense against pests and phytopathogenic microorganisms. In the last 20 years, several transgenic plants over-expressing SPIs have been produced and tested in order to achieve the increase of the resistance against pathogenic insects. Finally, in molecular farming, SPIs have been employed to minimize the proteolysis of recombinant proteins expressed in plants. The present review discusses the potential biotechnological applications of plant SPIs in the agriculture field.

Fabrication of silver chloride nanoparticles using a plant serine protease in combination with photoactivation and investigation of their biological activities.

Recently, the development of "green" methods for fabrication of silver nanoparticles (Ag-NPs) has been emphasized, in view of their environmental safety, feasibility, and low cost. In this study, a serine protease, EuP-82 from Euphorbia cf. lactea latex, was used to fabricate silver chloride nanoparticles (AgCl-NPs) in phosphate-buffered saline (pH 7.2), under the influence of visible light. The fabricated nanoparticles had a maximal surface plasmon resonance absorption peak at 435 nm. The size of the AgCl-NPs, estimated by scanning electron microscopy, was 57 ± 14.7 nm. Energy dispersive X-ray spectroscopy, X-ray absorption spectroscopy, and X-ray diffraction analysis confirmed that the fabricated Ag-NPs were of the AgCl type. The fabricated nanoparticles had antioxidant activity, scavenging DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals with IC50 of 204 ± 1.8 μg/mL. The fabricated AgCl-NPs had broad-spectrum in vitro antimicrobial activities, acting against the Gram-positive bacteria Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and Bacillus cereus, and the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. AgCl-NPs also showed antifungal activity against Candida albicans and C. tropicalis. In addition, AgCl-NPs showed antiprotozoal activity against Giardia lamblia, with IC50 202 ± 2.1 μg/mL. Based on the biological activities of the fabricated AgCl-NPs, they have the potential for widespread application in medicine and industry.

Heterologous expression and functional characterization of phytaspase, a caspase-like plant protease

Following the cloning and expression of tobacco (Nicotiana tabacum) phytaspase gene in Escherichia coli BL21, the recombinant protease was purified by affinity chromatography for further characterization. Circular dichroism (CD) spectroscopy and in silico analysis revealed structural similarities of recombinant phytaspase with other plant serine-proteases. Molecular docking studies showed favourable binding of synthetic peptide substrate for caspase 8 (Ac-VETD-AMC) to the reactive pocket of recombinant phytaspase indicating its potential in assessing functional activity of recombinant phytaspase. In silico findings were supported by caspase 8-like activity of purified phytaspase demonstrated in vitro. The Michaelis constant (KM) and specificity constant (kcat/KM) of phytaspase for hydrolyzing Ac-VETD-AMC were found to be 1.587μM and 4.67×103M−1min−1, respectively. Transient expression of phytaspase in lung epithelial adenocarcinoma cells (A549) resulted in reduced IC50 value of doxorubicin. This is the first report of functional expression of mature phytaspase in bacterial system as well as its transfection to sensitize A549 cells at lower doxorubicin concentration.

Fibrin(ogen)olytic and antiplatelet activities of a subtilisin-like protease from Solanum tuberosum (StSBTc-3).

Plant serine proteases have been widely used in food science and technology as well as in medicine. In this sense, several plant serine proteases have been proposed as potential anti-coagulants and anti-platelet agents. Previously, we have reported the purification and identification of a plant serine protease from Solanum tuberosum leaves. This potato enzyme, named as StSBTc-3, has a molecular weight of 72kDa and it was characterized as a subtilisin like protease. In this work we determine and characterize the biochemical and medicinal properties of StSBTc-3. Results obtained show that, like the reported to other plant serine proteases, StSBTc-3 is able to degrade all chains of human fibrinogen and to produces fibrin clot lysis in a dose dependent manner. The enzyme efficiently hydrolyzes β subunit followed by partially hydrolyzed α and γ subunits of human fibrinogen. Assays performed to determine StSBTc-3 substrate specificity using oxidized insulin β-chain as substrate, show seven cleavage sites: Asn3-Gln4; Cys7-Gly8; Glu13-Ala14; Leu15-Tyr16; Tyr16-Leu17; Arg22-Gly23 and Phe25-Tyr26, all of them were previously reported for other serine proteases with fibrinogenolytic activity. The maximum StSBTc-3 fibrinogenolytic activity was determined at pH 8.0 and at 37C. Additionally, we demonstrate that StSBTc-3 is able to inhibit platelet aggregation and is unable to exert cytotoxic activity on human erythrocytes invitro at all concentrations assayed. These results suggest that StSBTc-3 could be evaluated as a new agent to be used in the treatment of thromboembolic disorders such as strokes, pulmonary embolism and deep vein thrombosis.

Improved Detection of Remote Homologues Using Cascade PSI-BLAST: Influence of Neighbouring Protein Families on Sequence Coverage

BackgroundDevelopment of sensitive sequence search procedures for the detection of distant relationships between proteins at superfamily/fold level is still a big challenge. The intermediate sequence search approach is the most frequently employed manner of identifying remote homologues effectively. In this study, examination of serine proteases of prolyl oligopeptidase, rhomboid and subtilisin protein families were carried out using plant serine proteases as queries from two genomes including A. thaliana and O. sativa and 13 other families of unrelated folds to identify the distant homologues which could not be obtained using PSI-BLAST.Methodology/Principal FindingsWe have proposed to start with multiple queries of classical serine protease members to identify remote homologues in families, using a rigorous approach like Cascade PSI-BLAST. We found that classical sequence based approaches, like PSI-BLAST, showed very low sequence coverage in identifying plant serine proteases. The algorithm was applied on enriched sequence database of homologous domains and we obtained overall average coverage of 88% at family, 77% at superfamily or fold level along with specificity of ∼100% and Mathew’s correlation coefficient of 0.91. Similar approach was also implemented on 13 other protein families representing every structural class in SCOP database. Further investigation with statistical tests, like jackknifing, helped us to better understand the influence of neighbouring protein families.Conclusions/SignificanceOur study suggests that employment of multiple queries of a family for the Cascade PSI-BLAST searches is useful for predicting distant relationships effectively even at superfamily level. We have proposed a generalized strategy to cover all the distant members of a particular family using multiple query sequences. Our findings reveal that prior selection of sequences as query and the presence of neighbouring families can be important for covering the search space effectively in minimal computational time. This study also provides an understanding of the ‘bridging’ role of related families.

Crystal Structure of Cucumisin, a Subtilisin-Like Endoprotease from Cucumis melo L.

Cucumisin is a plant serine protease, isolated as an extracellular glycoprotein from the melon fruit Cucumis melo L. var. Prince. Cucumisin is composed of multiple domain modules, including catalytic, protease-associated, and fibronectin-III-like domains. The crystal structure of cucumisin was determined by the multiwavelength anomalous dispersion method and refined at 2.75Å resolution. A structural homology search indicated that the catalytic domain of cucumisin shares structural similarity with subtilisin and subtilisin-like fold enzymes. According to the Z-score, the highest structural similarity is with tomato subtilase 3 (SBT3), with an rmsd of 3.5Å for the entire region. The dimer formation mediated by the protease-associated domain in SBT3 is a distinctive structural characteristic of cucumisin. On the other hand, analytical ultracentrifugation indicated that cucumisin is mainly monomeric in solution. Although the locations of the amino acid residues composing the catalytic triad are well conserved between cucumisin and SBT3, a disulfide bond is uniquely located near the active site of cucumisin. The steric circumstances of the active site with this disulfide bond are distinct from those of SBT3, and it contributes to the substrate preference of cucumisin, especially at the P2 position. Among the plant serine proteases, the thermostability of cucumisin is higher than that of its structural homologue SBT3, as determined by their melting points. A structural comparison between cucumisin and SBT3 revealed that cucumisin possesses less surface area and shortened loop regions. Consequently, the higher thermostability of cucumisin is achieved by its more compact structure.

A novel serine protease with human fibrino(geno)lytic activities from Artocarpus heterophyllus latex

A protease was isolated and purified from Artocarpus heterophyllus (jackfruit) latex and designated as a 48-kDa antimicrobial protease (AMP48) in a previous publication. In this work, the enzyme was characterized for more biochemical and medicinal properties. Enzyme activity of AMP48 was strongly inhibited by phenylmethanesulfonyl fluoride and soybean trypsin inhibitor, indicating that the enzyme was a plant serine protease. The N-terminal amino acid sequences (A-Q-E-G-G-K-D-D-D-G-G) of AMP48 had no sequence similarity matches with any sequence databases of BLAST search and other plant serine protease. The secondary structure of this enzyme was composed of high α-helix (51%) and low β-sheet (9%). AMP48 had fibrinogenolytic activity with maximal activity between 55 and 60°C at pH 8. The enzyme efficiently hydrolyzed α followed by partially hydrolyzed β and γ subunits of human fibrinogen. In addition, the fibrinolytic activity was observed through the degradation products by SDS-PAGE and emphasized its activity by monitoring the alteration of secondary structure of fibrin clot after enzyme digestion using ATR-FTIR spectroscopy. This study presented the potential role to use AMP48 as antithrombotic for treatment thromboembolic disorders such as strokes, pulmonary emboli and deep vein thrombosis.

Serine protease inhibitors in plants: nature’s arsenal crafted for insect predators

Plant serine protease inhibitors are defense proteins crafted by nature for inhibiting serine proteases. Use of eco-friendly, sustainable and effective protein molecules which could halt or slow down metabolism of nutrients in pest would be a pragmatic approach in insect pest management of crops. The host-pest complexes that we observe in nature are evolutionary dynamic and inter-depend on other defense mechanisms and interactions of other pests or more generally speaking symbionts with the same host. Insects have co-evolved and adapted simultaneously, which makes it necessary to investigate serine protease inhibitors in non-host plants. Such novel serine protease inhibitors are versatile candidates with vast potential to overcome the host inhibitor-insensitive proteases. In a nutshell exploring and crafting plant serine proteinase inhibitors (PIs) for controlling pests effectively must go on. Non-host PI seems to be a better choice for coevolved insensitive proteases. Transgenic plants expressing wound inducible chimaeric PIs may be an outstanding approach to check wide spectrum of gut proteinases and overcome the phenomenon of resistance development. Thus, this article focuses on an entire array of plant serine protease inhibitors that have been explored in the past decade, their mode of action and biological implications as well as applications.

The multiple functions of plant serine protease inhibitors

Plant protease inhibitors (PIs) are a diverse group of proteins which have been intensely investigated due to their potential function in protecting plants against herbivorous insects by inhibiting digestive proteases. Although this mechanism has been well documented for a number of single PIs and their target enzymes, whether this mechanism protects plants in nature remains unclear. Moreover, many plants express a number of different PIs and it was unknown if these proteins work synergistically as defenses or if they also have other functions. We recently identified four serine PIs (SPI) of Solanum nigrum and demonstrated that they differ substantially in substrate specificity, accumulation patterns, and their effect against different natural herbivorous insects in field- and glasshouse experiments. These differences suggest that SPIs have at least partially diversified to provide protection against different attackers. Although we could not detect effects on plant development or growth when silencing SPIs, gene- and tissue-specific expression patterns suggest multiple functions in generative tissues, including a possible involvement in development.

Purification and biochemical characterisation of a novel protease streblin

A serine protease, streblin, was purified 4.6-fold with 75% recovery from the latex of a plant, Streblus asper. This is the first report of identification and purification of a serine protease from the genus Streblus of the Moraceae family. Streblin, has a molecular mass of 64 kDa and the extinction coefficient ( ϵ 280 1 % ) is 5.29. Streblin is a basic protein with pI value of 9.2 that acts optimally at pH 9.0; such optimum activity at high pH has not been reported for most of the isolated plant serine proteases and the enzyme is stable over a wide range of pH (3.0–12.5). The enzyme is also thermostable, retaining complete activity at 15–85 °C and acts optimally at 65 °C. Furthermore, it is highly stable in the presence of various denaturants in which SDS resistance is the most striking property of the purified enzyme. Streblin strongly coagulated skimmed milk. Easy availability of the latex, simple purification procedures, high stability of streblin against pH, or autodigestion, and under various conditions make the enzyme a good system for exploring the biophysical chemistry of proteases. In addition to its high milk-clotting ability, it could be used in the cheese industry, as well as other food and biotechnological industries.

Functional Analysis of the Cucumisin Propeptide as a Potent Inhibitor of Its Mature Enzyme

Cucumisin is a subtilisin-like serine protease (subtilase) that is found in the juice of melon fruits (Cucumis melo L.). It is synthesized as a preproprotein consisting of a signal peptide, NH(2)-terminal propeptide, and 67-kDa protease domain. We investigated the role of this propeptide (88 residues) in the cucumisin precursor. Complementary DNAs encoding the propeptides of cucumisin, two other plant subtilases (Arabidopsis ARA12 and rice RSP1), and bacterial subtilisin E were expressed in Escherichia coli independently of their mature enzymes. The cucumisin propeptide strongly inhibited cucumisin in a competitive manner with a K(i) value of 6.2 ± 0.55 nm. Interestingly, cucumisin was also strongly inhibited by ARA12 and RSP1 propeptides but not by the subtilisin E propeptide. In contrast, the propeptides of cucumisin, ARA12, and RSP1 did not inhibit subtilisin. Deletion analysis clearly showed that two hydrophobic regions, Asn(32)-Met(38) and Gly(97)-Leu(103), in the cucumisin propeptide were important for its inhibitory activity. Site-directed mutagenesis also confirmed the role of a Val(36)-centerd hydrophobic cluster within the Asn(32)-Met(38) region in cucumisin inhibition. Circular dichroism spectroscopy revealed that the cucumisin propeptide had a secondary structure without a cognate protease domain and that the thermal unfolding of the propeptide at 90 °C was only partial and reversible. A tripeptide, Ile(35)-Val(36)-Tyr(37), in the Asn(32)-Met(38) region was thought to contribute toward the formation of a proper secondary structure necessary for cucumisin inhibition. This is the first report on the function and structural information of the propeptide of a plant serine protease.

CHARACTERIZATION OF THREE NOVEL ALKALINE SERINE PROTEASES FROM TOMATO (LYCOPERSICUM ESCULENTUM MILL.) FRUIT AND THEIR POTENTIAL APPLICATION

In order to quest new potential plant protease and make it industrially applicable and cost-effective, three novel proteases were detected in tomato (Lycopersicum esculentum Mill.) fruit by sodium dodecyl sulfate (SDS)–gelatin–polyacrylamide gel electrophoresis. All three proteases were isolated and partially purified by heat treatment (70C, 5 min) and ethanol precipitation (0–33%, v/v), with ninefold increase in specific activity and 31.6% recovery. These proteases exhibited the highest specificity for gelatin substrate and were indentified as serine protease. The optimum pH and temperature of proteolytic activity were pH 8–10 and 37–60C, respectively. They were stable at high pH (up to 12) and temperature (up to 70C) and in the presence of 20 mM metal ions (Na+, Ba2+, Ca2+, Mg2+, Mn2+), surfactants (SDS and 80 mM Triton X-100) and denaturants (urea and dithiothreitol). These results suggested that proteases from tomato fruit had potential application in food, detergent, tannery and medical industries. PRACTICAL APPLICATIONS Plant cysteine proteases such as papain, bromelain and ficin are extensively applied in many industries. Recently, several plant serine proteases, which were more stable under demanding conditions than cysteine proteases, were exploited for industrial applications. Tomato is one of the highest produced and consumed vegetables in the worldwide. Alkaline serine proteases from tomato fruit in this paper were stable at high pH and temperature and in the presence of metal ions and oxidation. Enzyme activity against a wide variety of nature protein substrates indicated that they would be investigated for a range of potential commercial applications especially for gelatin and casein hydrolysis in the food processing industry as well as for the dissolution of casein and bovine hemoglobin in detergent industry.

Isolation and partial sequence of a Kunitz-type elastase specific inhibitor from marama bean (Tylosema esculentum)

An isolation procedure utilizing ammonium sulfate fractionation and affinity chromatography was used to purify an elastase inhibitor present in large amounts in marama beans (Tylosema esculentum). The protein appeared to be heterogeneous due to carbohydrate differences, demonstrating two bands on SDS gels with molecular weights of 17.8 kDa and 20 kDa. Partial sequence, derived from mass spectrometry, indicated that the protein is a Kunitz-type inhibitor distinct from other known plant serine protease inhibitors. The marama bean inhibitor is specific for elastase, with very low Ki for both pancreatic and neutrophil elastase. The quantity of elastase inhibitor present in marama beans is many times greater than in soybean or any other bean or nut source reported to date. This raises the question of why a bean found in an arid corner of the Kalahari Desert would be so rich in a very potent elastase inhibitor.

Carnein, a Serine Protease from Noxious Plant Weed Ipomoea carnea (Morning Glory)

A new serine protease from the latex of Ipomoea carnea spp. fistulosa (Morning glory), belonging to the Convolvulaceae family, was purified to homogeneity by ammonium sulfate fractionation followed by cation exchange chromatography. The enzyme, named carnein, has a molecular mass of 80.24 kDa (matrix-assisted laser desorption/ionization time-of-flight) and an isoelectric point of pH 5.6. The pH and temperature optima for proteolytic activity were 6.5 and 65 degrees C, respectively. The extinction coefficient (epsilon2801%) of the enzyme was estimated as 37.12, and the protein molecule consists of 35 tryptophan, 76 tyrosine, and seven cysteine residues. The effect of several inhibitors such as iodoacetic acid, diisopropylfluorophosphate, phenyl-methanesulfonyl fluoride, chymostatin, soybean trypsin inhibitor, HgCl2, 3S-3-(N-{(S)-1-[N-(4-guanidinobutyl)carbamoyl]3-ethylbutyl}carbamoyl)oxirane-2-carboxylic acid, N-ethyl maleimide, ethylene glycol-bis(alpha-amino ethyl ether)tetraacetic acid, ethylenediamminetetraacetic acid, and o-phenonthroline indicates that carnein belongs to the family of serine proteases. The enzyme is not prone to autolysis even at very low concentrations. The N-terminal sequence of carnein (T-T-H-S-P-E-F-L-G-L-A-E-S-S-G-L-X-P-N-S) exhibited considerable similarity to those of other plant serine proteases; the highest similarity was with alnus AG12, one of the subtilase family endopepetidases.