Clinical study of anti-snake venom blockade in the treatment of local tissue necrosis caused by Chinese cobra (Naja atra) bites.

OBJECTIVE: This study aimed to evaluate the clinical therapeutic efficacy of anti-snake venom serum blockade in treating local tissue necrosis caused by Chinese cobra (Naja atra) bites. METHODS: Patients bitten by a Chinese cobra (Naja atra) (n = 50) that met the inclusion criteria were randomly divided into two groups: the experimental group (n = 25) and the control group (n = 25). The experimental group received regular as well as anti-snake venom serum blocking treatment, whereas regular treatment plus chymotrypsin blocking therapy was given to the control group. The necrotic volumes around snake wounds in these groups were detected on the first, third and seventh days. On the third day of treatment, some local tissues in the wounds were randomly selected for pathological biopsy, and the necrosis volume of the local tissue was observed. Furthermore, the amount of time required for wound healing was recorded. RESULTS: On the third and seventh days post-treatment, the necrotic volume of the wound of the experimental group was much smaller than that of the control group, and the experimental group's wound healing time was shorter than that of the control group (all p < 0.05). Moreover, the pathological biopsies taken from the control group showed nuclear pyknosis, fragmentation, sparse nuclear density, and blurred edges, and the degree of necrosis was much higher than that of the experimental group. CONCLUSIONS: Anti-snake venom blocking therapy is a new and improved therapy with good clinical effect on local tissue necrosis caused by Chinese cobra bites; moreover, it is superior to conventional chymotrypsin blocking therapy in the treatment of cobra bites. It can better neutralize and prevent the spread of the toxin, reduce tissue necrosis, and shorten the course of the disease by promoting healing of the wound. Furthermore, this treatment plan is also applicable to wound necrosis caused by other snake toxins, such as tissue necrosis caused by elapidae and viper families. CLINICAL TRIAL REGISTRATION: This trial is registered in the Chinese Clinical Trial Registry, a primary registry of International Clinical Trial Registry Platform, World Health Organization (Registration No. ChiCTR2200059070; trial URL:http://www.chictr.org.cn/edit.aspx?pid=134353&htm=4).

A human-blood-derived microRNA facilitates flavivirus infection in fed mosquitoes.

Hematophagous arthropods, such as mosquitoes, naturally carry and transmit hundreds of arboviruses to humans. Blood meal is a predominant physical interface that shapes cross-species communications among humans, bloodsuckers, and arboviruses. Here, we identify a human-blood-derived microRNA, hsa-miR-150-5p, that interferes with a mosquito antiviral system to facilitate flavivirus infection and transmission. hsa-miR-150-5p is acquired with a blood meal into the mosquito hemocoel and persists for a prolonged time there. The agomir of hsa-miR-150-5p enhances, whereas the antagomir represses flaviviral infection in mosquitoes and transmission from mice to mosquitoes. Mechanistic studies indicate that hsa-miR-150-5p hijacks the mosquito Argonaute-1-mediated RNA interference system to suppress the expression of some chymotrypsins with potent virucidal activity. Mosquito chymotrypsins are essential for resisting systemic flavivirus infection in hemocoel tissues. Chymotrypsin homologs potentially targeted by miR-150-5p are also found in other hematophagous arthropods, demonstrating a conserved miR-150-5p-mediated cross-species RNAi mechanism that might determine flaviviral transmissibility in nature.

A 584 bp deletion in CTRB2 inhibits chymotrypsin B2 activity and secretion and confers risk of pancreatic cancer.

Genome-wide association studies (GWASs) have discovered 20 risk loci in the human genome where germline variants associate with risk of pancreatic ductal adenocarcinoma (PDAC) in populations of European ancestry. Here, we fine-mapped one such locus on chr16q23.1 (rs72802365, p = 2.51 × 10-17, OR = 1.36, 95% CI = 1.31-1.40) and identified colocalization (PP = 0.87) with aberrant exon 5-7 CTRB2 splicing in pancreatic tissues (pGTEx = 1.40 × 10-69, ßGTEx = 1.99; pLTG = 1.02 × 10-30, ßLTG = 1.99). Imputation of a 584 bp structural variant overlapping exon 6 of CTRB2 into the GWAS datasets resulted in a highly significant association with pancreatic cancer risk (p = 2.83 × 10-16, OR = 1.36, 95% CI = 1.31-1.42), indicating that it may underlie this signal. Exon skipping attributable to the deletion (risk) allele introduces a premature stop codon in exon 7 of CTRB2, yielding a truncated chymotrypsinogen B2 protein that lacks chymotrypsin activity, is poorly secreted, and accumulates intracellularly in the endoplasmic reticulum (ER). We propose that intracellular accumulation of a nonfunctional chymotrypsinogen B2 protein leads to ER stress and pancreatic inflammation, which may explain the increased pancreatic cancer risk in carriers of CTRB2 exon 6 deletion alleles.

Localization of the proteinase inhibitor activity in the fish cestode Eubothrium rugosum.

The mechanisms enabling fish tapeworms to avoid proteolytic attacks by digestive enzymes of their fish host have been studied in less detail compared with mammalian cestodes. This study aimed to assess the inhibitory ability towards trypsin and chymotrypsin in Eubothrium rugosum, an intestinal parasite of burbot Lota lota, and establish its localization in the tapeworm. To this end, the worms were treated with Triton X-100 followed by differential centrifugation to isolate the tegumental brush border membrane. The protease inhibitory abilities of the worms were mostly determined by their excretory/secretory products released into the incubation medium. These inhibitory abilities proved to be linked mainly with the brush border fractions. Notably, the per cent inhibition of both studied digestive enzymes (trypsin and chymotrypsin) hardly depended on the duration of the parasite exposure in the incubation medium, probably due to intermittent glycocalyx renewal. Improved knowledge on functions of the excretory/secretory proteins produced by fish tapeworms may contribute to a better understanding of host-parasite relations and development of new approaches to the treatment and prevention of diseases caused by pathogenic helminths.

Reversible Photoswitchable Inhibitors Generate Ultrasensitivity in Out-of-Equilibrium Enzymatic Reactions.

Ultrasensitivity is a ubiquitous emergent property of biochemical reaction networks. The design and construction of synthetic reaction networks exhibiting ultrasensitivity has been challenging, but would greatly expand the potential properties of life-like materials. Herein, we exploit a general and modular strategy to reversibly regulate the activity of enzymes using light and show how ultrasensitivity arises in simple out-of-equilibrium enzymatic systems upon incorporation of reversible photoswitchable inhibitors (PIs). Utilizing a chromophore/warhead strategy, PIs of the protease α-chymotrypsin were synthesized, which led to the discovery of inhibitors with large differences in inhibition constants (Ki) for the different photoisomers. A microfluidic flow setup was used to study enzymatic reactions under out-of-equilibrium conditions by continuous addition and removal of reagents. Upon irradiation of the continuously stirred tank reactor with different light pulse sequences, i.e., varying the pulse duration or frequency of UV and blue light irradiation, reversible switching between photoisomers resulted in ultrasensitive responses in enzymatic activity as well as frequency filtering of input signals. This general and modular strategy enables reversible and tunable control over the kinetic rates of individual enzyme-catalyzed reactions and makes a programmable linkage of enzymes to a wide range of network topologies feasible.

Isolation and structure determination of new chymotrypsin inhibitory peptides streptopeptolins B and C.

New chymotrypsin inhibitory peptides named streptopeptolins B and C were isolated from Streptomyces olivochromogenes. Structures of streptopeptolins B and C were determined to be cyclic depsipeptides possessing 3-amino-6-hydroxy-2-piperidone unit by interpretation of NMR spectra and ESI-MS. Streptopeptolins B and C showed inhibitory activities to chymotrypsin with IC50 of 8.0 and 12.0 µg/mL, respectively.

Identification and Target-Modification of SL-BBI: A Novel Bowman-Birk Type Trypsin Inhibitor from Sylvirana latouchii.

The peptides from the ranacyclin family share similar active disulphide loop with plant-derived Bowman-Birk type inhibitors, some of which have the dual activities of trypsin inhibition and antimicrobial. Herein, a novel Bowman-Birk type trypsin inhibitor of the ranacyclin family was identified from the skin secretion of broad-folded frog (Sylvirana latouchii) by molecular cloning method and named as SL-BBI. After chemical synthesis, it was proved to be a potent inhibitor of trypsin with a Ki value of 230.5 nM and showed weak antimicrobial activity against tested microorganisms. Modified analogue K-SL maintains the original inhibitory activity with a Ki value of 77.27 nM while enhancing the antimicrobial activity. After the substitution of active P1 site to phenylalanine and P2' site to isoleucine, F-SL regenerated its inhibitory activity on chymotrypsin with a Ki value of 309.3 nM and exhibited antiproliferative effects on PC-3, MCF-7 and a series of non-small cell lung cancer cell lines without cell membrane damage. The affinity of F-SL for the ß subunits in the yeast 20S proteasome showed by molecular docking simulations enriched the understanding of the possible action mode of Bowman-Birk type inhibitors. Further mechanistic studies have shown that F-SL can activate caspase 3/7 in H157 cells and induce apoptosis, which means it has the potential to become an anticancer agent.

Serpins in Fasciola hepatica: insights into host-parasite interactions.

Protease inhibitors play crucial roles in parasite development and survival, modulating the immune responses of their vertebrate hosts. Members of the serpin family are irreversible inhibitors of serine proteases and regulate systems related to defence against parasites. Limited information is currently available on protease inhibitors from the liver fluke Fasciola hepatica. In this study, we characterised four serpins from F. hepatica (FhS-1-FhS-4). Biochemical characterisation revealed that recombinant FhS-2 (rFhS) inhibits the activity of human neutrophil cathepsin G, while rFhS-4 inhibits the activity of bovine pancreatic chymotrypsin and cathepsin G. Consistent with inhibitor function profiling data, rFhS-4 inhibited cathepsin G-activated platelet aggregation in a dose-responsive manner.Similar to other serpins, rFhS2 and rFhS-4 bind to heparin with high affinity. Tissue localisation demonstrated that these serpins have different spatial distributions. FhS-2 is localised in the ovary, while FhS-4 was found in gut cells. Both of them co-localised in the spines within the tegument. These findings provide the basis for study of functional roles of these proteins as part of an immune evasion mechanism in the adult fluke, and in protection of eggs to ensure parasite life cycle continuity. Further understanding of serpins from the liver fluke may lead to the discovery of novel anti-parasitic interventions.

Partial Characterization of Protease Inhibitors of Ulva ohnoi and Their Effect on Digestive Proteases of Marine Fish.

This piece of research evaluates the presence of protease inhibitors in the macroalga Ulva ohnoi and provides an initial overview of their mode of action. The ability of Ulva protease inhibitors to inhibit digestive proteases of three marine fish species, as well as their capacity to hamper the hydrolysis of a reference protein by those fish proteases, were assessed. In addition, thermal stability and the mode of inhibition on trypsin and chymotrypsin were also studied. Dose-response inhibition curves and in vitro protein hydrolysis assays revealed a noticeable inhibition of fish enzymes when Ulva concentration increased in the assay. The thermal treatment of Ulva reduced markedly the inhibitory effect on fish digestive protease. Finally, Lineweaver-Burk plots indicated that trypsin and chymotrypsin inhibition consisted of a mixed-type inhibition mechanism in which the inhibitory effect depends on Ulva concentration. Overall, the results confirmed the presence of protease inhibitors in Ulva, though heat treatment was enough for inactivating these compounds.

Bromo-spiroisoxazoline Alkaloids, Including an Isoserine Peptide, from the Caribbean Marine Sponge Aplysina lacunosa.

Three new bromotyrosine spiroisoxazoline alkaloids, lacunosins A and B (1 and 2) and desaminopurealin (3), were isolated from a MeOH extract of the marine sponge Aplysina lacunosa that showed modest α-chymotrypsin inhibitory activity. The structures of 1-3 share the spirocyclohexadienyl-isoxazoline ring system found in purealidin-R and several other Verongid sponge secondary metabolites. Compounds 1 and 2 are coupled to a glycine and an isoserine methyl ester, respectively. Alkaloid 3 is linked, contiguously, to an O-1-aminopropyl 3,5-dibromotyrosyl ether and, finally, to histamine through an amide bond. The planar structures of all three compounds were obtained from analysis of MS and 1D and 2D NMR data. The absolute configuration of the SIO unit of 1-3 was assigned by electronic circular dichroism (ECD). The isoserine amino acid residue in 2 was found to be a 1:1 mixture of epimers using a new Marfey's type reagent, derived from Trp-NH2. Allylic O-naphthoylation of the SIO subunit enhances the ECD spectrum of SIOs and improves discrimination of enantiomorphs. A unifying hypothesis is proposed that links the biosynthesis of several of the new compounds with previously reported analogues.

Amblyomma americanum serpin 41 (AAS41) inhibits inflammation by targeting chymase and chymotrypsin.

Ticks inject serine protease inhibitors (serpins) into their feeding sites to evade serine protease-mediated host defenses against tick-feeding. This study describes two highly identitical (97%) but functionally different Amblyomma americanum tick saliva serpins (AAS41 and 46) that are secreted at the inception of tick-feeding. We show that AAS41, which encodes a leucine at the P1 site inhibits inflammation system proteases: chymase (SI = 3.23, Ka = 5.6 ± 3.7X103M-1 s-1) and α-chymotrypsin (SI = 3.18, Ka = 1.6 ± 4.1X104M-1 s-1), while AAS46, which encodes threonine has no inhibitory activity. Similary, rAAS41 inhibits rMCP-1 purified from rat peritonuem derived mast cells. Consistently, rAAS41 inhibits chymase-mediated inflammation induced by compound 48/80 in rat paw edema and vascular permeability models. Native AAS41/46 proteins are among tick saliva immunogens that provoke anti-tick immunity in repeatedly infested animals as revealed by specific reactivity with tick immune sera. Of significance, native AAS41/46 play critical tick-feeding functions in that RNAi-mediated silencing caused ticks to ingest significantly less blood. Importantly, monospecific antibodies to rAAS41 blocked inhibitory functions of rAAS41, suggesting potential for design of vaccine antigens that provokes immunity to neutralize functions of this protein at the tick-feeding site. We discuss our findings with reference to tick-feeding physiology and discovery of effective tick vaccine antigens.

Inhibition of α-chymotrypsin by pristine single-wall carbon nanotubes: Clogging up the active site.

The preferred spatial orientation of single-wall carbon nanotubes (SWCNTs) in their interaction with enzymes determines their behavior either as nano-supports or as inhibitors. α -chymotrypsin (α-CT) is considered a serine protease model for studying nanomaterial/proteases interactions. The interaction of α-CT with pristine single-wall carbon nanotubes is still unknown. Here α-CT/SWCNT hybrids are synthesized and characterized. Spectroscopic, microscopic and kinetic measurements, coupled to molecular dynamics simulations, provide a detailed description of the interaction between α-CT and SWCNTs. The SWCNT binding pocket was unambiguously identified. A perfect match is observed with the crevice structure of the α-CT substrate binding pocket. The activity of α-CT, upon SWCNT binding, is dramatically reduced, as expected by the interaction of the SWCNT in the active site of the protein. π-π stacking between aromatic residues and the conjugated surface of SWCNT governs α-CT/SWCNT interactions. An important role in the bonding appears also for purely hydrophobic residues and with residues able to establish surfactant-like interactions. The secondary structure of α-CT and the catalytic triad structure are not perturbed by the complex formation, on the contrary the volume of the substrate binding pocket is strongly reduced by SWCNT binding because SWCNT occupies the α-CT substrate binding site, clogging the active site.

TLC-Bioautography as a fast and cheap screening method for the detection of α-chymotrypsin inhibitors in crude plant extracts.

TLC-Bioautography is a fast and effective method for assessing the inhibitory effect of compounds present in plant extracts against microbial species. However, this method has a hidden, currently underutilized potential for evaluating the presence of inhibitory compounds against selected enzymes. The aim of this work was to design a functional TLC-Bioautography method for the evaluation of protease inhibitors present in plant extracts. The method is based on the hydrolysis of Nα-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BApNA) by α-chymotrypsin as a representative serine protease to produce coloured para-nitroaniline (pNA). Derivatization of pNA with both sodium nitrite and N-(1-naphthyl) ethylenediamine (NPED) leads to the formation of a pink azo dye. This step improves the resolution of active compounds on the chromatogram, which appear as light spots on a pink background. The developed method was tested for the analysis of protease inhibitors in different plant materials such as grape pomace from Vitis vinifera, Picea abies bark, Hippophae rhamnoides berries, Hordeum sativum bran, Triticum aestivum bran and Avena sativa bran. Plant extracts, which could not be analysed by a commonly used spectrophotometric method due to interference, were assessed by this method.

Cucurbit[7]uril-Mediated 2D Single-Layer Hybrid Frameworks Assembled by Tetraphenylethene and Polyoxometalate toward Modulation of the α-Chymotrypsin Activity.

Construction of large-scale single-layer two-dimensional (2D) frameworks in water is significant due to their utilities in various fields. Utilizing macrocycle-mediated supramolecular self-assembly represents a promising approach; however, challenges still remain in their practical preparation. Here, we exploited a two-step supramolecular strategy to build 2D organic-inorganic hybrid frameworks at a micrometer scale in water. Taking advantage of the high binding affinity to cucurbit[7]uril (CB[7]), mono-quaternary ammonium tetraphenylethene (MQATPE) derivatives were first included with CB[7] to form a 1:1 complex (MQATPE@CB[7]). Then, just mixing the complex with anionic polyoxometalate Na9[EuW10O36]·32H2O (denoted as Eu-POM) in a 3:1 molar ratio leads to the formation of single-layer 2D films with tens of micrometers via electrostatic and π-π stacking interactions. The most unique feature of this strategy is that the steric effect imposed by CB[7] would not only lead the modules to adopt a periodic hexagonal assembly but also forbid stacking between layers through comparison with the merely multilayered 2D nanosheets self-assembled by MQATPE/Eu-POM. Interestingly, the charge interactions between MQATPE and Eu-POM would lead to the aggregation-induced emission (AIE) fluorescence of MQATPE, and white light emission could be obtained through the simple regulation of the contents of Eu-POM and MQATPE. Furthermore, due to the high surface areas and more accessible active sites, the single-layer films can act as an effective enzyme inhibitor to modulate the activity of α-chymotrypsin (ChT). These findings suggest a simple but universal approach for single-layer hybrid materials, which may hold promise for practical applications in photophysical and biomedical fields.

Expression and purification of recombinant human serpin B1 yields novel molecules with altered protease inhibitory activities: Functional implications.

Serpin B1 regulates the innate immune system by inhibiting serine and cysteine proteases that control programmed cell death and proliferation pathways. To provide recombinant human proteins for in vitro and in vivo studies we expressed and purified wild-type human serpin B1 and a C344A variant in the yeast S. cerevisiae. Both proteins expressed well and inhibited elastase and chymotrypsin. However, purification of wild-type serpin B1 in the absence of a reducing agent resulted in the specific loss of elastase - but not chymotrypsin - inhibition, concomitant with the formation of two higher molecular weight forms of the protein - a modified monomer and a dimer created via an intermolecular disulfide bond formed between C344 in respective serpin B1 monomers. In contrast to fully reduced serpin B1, both modified forms were good elastase substrates and catalytically cleaved at multiple adjacent sites within the reactive site loop. In contrast, purification of the C344A variant in the absence of a reducing agent yielded only one form of the protein which retained elastase and chymotrypsin inhibitory properties when purified. Furthermore, the elastase inhibitory activity of wild-type serpin B1, but not the C344A variant, was sensitive to oxidation. Thus, wild-type human serpin B1 should be formulated with a pharmaceutically acceptable reducing agent to protect C344 against post-translational oxidative modifications. Alternatively, the C344A variant of this protein may prove to be a suitable drug development candidate. These findings also suggest that inactivation of serpin B1 by oxidation may have a physiological role to play during inflammation.

Introduction of Bifunctionality into the Multidomain Architecture of the ω-Ester-Containing Peptide Plesiocin.

The modular biosynthetic pathway of ribosomally synthesized and post-translationally modified peptides (RiPPs) enhances their engineering potential for exploring new structures and biological functions. The ω-ester-containing peptides (OEPs), a subfamily of RiPPs, have distinct side-to-side ester or amide linkages and frequently present more than one macrocyclic domain in a "beads-on-a-string" structure. In an effort to improve the engineering potential of RiPPs, we present here the idea that the multidomain architecture of an OEP, plesiocin, can be exploited to create a bifunctional modified peptide. Characterization of plesiocin variants revealed that strong chymotrypsin inhibition relies on the bicyclic structure of the domain in which a leucine residue in the hairpin loop functions as a specificity determinant. Four domains of plesiocin promote simultaneous binding of multiple enzymes, where the C-terminal domain binds chymotrypsin most efficiently. Using this information, we successfully engineered a plesiocin variant in which two different domains inhibit chymotrypsin and trypsin. This result suggests that the multidomain architecture of OEPs is a useful platform for engineering multifunctional hybrid RiPPs.

A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa.

Serine proteases play pivotal roles in normal physiology and a spectrum of patho-physiological processes. Accordingly, there is considerable interest in the discovery and design of potent serine protease inhibitors for therapeutic applications. This led to concerted efforts to discover versatile and robust molecular scaffolds for inhibitor design. This investigation is a bioprospecting study that aims to isolate and identify protease inhibitors from the cnidarian Actinia tenebrosa. The study isolated two Kunitz-type protease inhibitors with very similar sequences but quite divergent inhibitory potencies when assayed against bovine trypsin, chymostrypsin, and a selection of human sequence-related peptidases. Homology modeling and molecular dynamics simulations of these inhibitors in complex with their targets were carried out and, collectively, these methodologies enabled the definition of a versatile scaffold for inhibitor design. Thermal denaturation studies showed that the inhibitors were remarkably robust. To gain a fine-grained map of the residues responsible for this stability, we conducted in silico alanine scanning and quantified individual residue contributions to the inhibitor's stability. Sequences of these inhibitors were then used to search for Kunitz homologs in an A. tenebrosa transcriptome library, resulting in the discovery of a further 14 related sequences. Consensus analysis of these variants identified a rich molecular diversity of Kunitz domains and expanded the palette of potential residue substitutions for rational inhibitor design using this domain.

Synthesis of Oligomeric and Monomeric Functionalized Graphene Oxides and a Comparison of Their Abilities to Perform as Protein Ligands and Enzyme Inhibitors.

Graphene oxide (GO) is a versatile, monomolecular layered nanomaterial that possesses various oxygen-containing functionality on its large surface. These characteristics allow GO to interact with a variety of materials and to be applied towards a number of areas. The strength and selectivity of these interactions can be improved significantly through further functionalization. In this paper, we describe the functionalization of GO and its application as a protein ligand and an enzyme inhibitor. The work reported in this paper details how chymotrypsin inhibition can be improved using GO functionalized with a monomeric and oligomer layer of tyrosine. The results indicated that the mono- and oligo-functionalized systems performed extremely well, with Ki values nearly four times better than GO alone. Our original premise was that the oligomeric system would bind better because of the length of the oligomeric arms and potential for a high degree of flexibility. However, the results clearly showed that the shorter monomeric system was the better ligand/inhibitor. This was due to weaker intramolecular interactions between the aromatic side chains of tyrosine and the aromatic surface of GO. Although these are possible for both systems, they are cooperative and therefore stronger for the oligomeric functionalized GO. As such, the protein must compete and overcome these cooperative intramolecular interactions before it can bind to the functionalized GO, whereas the tyrosines on the surface of the monomeric system interact with the surface of GO through a significantly weaker monovalent interaction, but interact cooperatively with the protein surface.

Coix lacryma-jobi chymotrypsin inhibitor displays antifungal activity.

A novel chymotrypsin inhibitor, named ClCI, was purified from coix seed (Coix lacryma-jobi L.) by aqueous two-phase extraction, chymotrypsin-Sepharose 4B affinity chromatography and centrifugal ultrafiltration. ClCI was a 7.9 kDa competitive inhibitor with pI 6.54. The inhibition constants (Ki) for bovine pancreatic chymotrypsin and bacterial subtilisin were 1.27 × 10-10 M and 1.57 × 10-9 M respectively. ClCI had no inhibitory activity against bovine trypsin and porcine elastase. ClCI had wide pH stability and good heat resistance. It can maintain >90% inhibition activity against chymotrypsin at 20-80 °C for 1 h. The primary structure of ClCI was highly similar (57%-92%) to those of several inhibitors belonging to the Gramineae crop potato protease inhibitor- I superfamily and showed the typical sequence motif of the protease inhibitor of the seed storage protein group. ClCI (12.5 mg) inhibited mycelial growth of the phytopathogenic fungi Mycosphaerella melonis, Helminthosporium turcicum, Alternaria solani, Phytophthora capsici, Isariopsis griseola, and Colletotrichum gloeosporioides, and caused 89% inhibition of the proteases from spore germination of plant-pathogenic fungi. The results of the present study indicate that ClCI had biotechnological potential as an alternative agent to combat the important phytopathogenic fungi.

Light scattering determination of the stoichiometry for protease-potato serine protease inhibitor complexes.

The interaction between pancreatic proteases and a serine protease inhibitor purified from potato tubers was investigated by chromatography-coupled light scattering measurements. The molar mass distribution in the chromatogram was compared to theoretical values calculated for the different possible combinations of complexes and free components by three different approaches, namely section analyses of the chromatograms, full mass average determination and mass distribution analysis. This revealed that the inhibitor was able to bind trypsin in a 2:1 complex, whereas the data for chymotrypsin clearly showed a limitation to 1:1 complex regardless of the molar ratio in the injected samples. The same experiment carried out with elastase and the potato inhibitor gave only weak indications of complex formation under the conditions used.