Gel Filtration HPLC Research Articles

Tendency for oxidation of annelid hemoglobin at alkaline pH and dissociated states probed by redox titration

The redox titration of extracellular hemoglobin of Glossoscolex paulistus (Annelidea) was investigated in different pH conditions and after dissociation induced by pressure. Oxidation increased with increasing pH, as shown by the reduced amount of ferricyanide necessary for the oxidation of hemoglobin. This behavior was the opposite of that of vertebrate hemoglobins. The potential of half oxidation ( E 1 / 2 ) changed from − 65.3 to + 146.8 mV when the pH increased from 4.50 to 8.75. The functional properties indicated a reduction in the log P 50 from 1.28 to 0.28 in this pH range. The dissociation at alkaline pH or induced by high pressure, confirmed by HPLC gel filtration, suggested that disassembly of the hemoglobin could be involved in the increased potential for oxidation. These results suggest that the high stability and prolonged lifetime common to invertebrate hemoglobins is related to their low tendency to oxidize at acidic pH, in contrast to vertebrate hemoglobins.

A Recombinant Precursor of the Mustard Allergen Sin a 1 Retains the Biochemical and Immunological Features of the Heterodimeric Native Protein

Background: Mustard has been an important cause of food allergy of increasing incidence in the last years. Sin a 1, a storage 2S albumin, is the most relevant allergen from this spice. Methods:Pichia pastoris has been used as host for the recombinant production of the precursor form of Sin a 1 (rproSin a 1). rproSin a 1 was purified to homogeneity by three chromatographic steps: gel filtration, anion exchange and reverse-phase HPLC. Molecular characterization was performed using Edman degradation, mass spectrometry, amino acid composition, and circular dichroism. Immunological properties were analyzed by immunoblotting, ELISA, and ELISA inhibition experiments. Results: We overexpressed rproSin a 1 as a single polypeptide with both large and small chains linked by an internal processed fragment at high yield. The purified rproSin a 1 (>95%) was obtained as a monomeric and soluble protein. rproSin a 1 showed equivalent structural and immunological properties to natural heterodimeric Sin a 1 allergen. rproSin a 1 was recognized by 75% of the patients allergic to mustard. The inhibitory capacity of rproSin a 1 to the total allergenicity of mustard extracts varied from 13 to 83% in different patients, with a mean value of 54%. Conclusions: rproSin a 1 is a good candidate to replace natural allergen in diagnosis protocols of mustard allergy. P. pastoris has been demonstrated to be a suitable expression system for the production of allergenic derivates of Sin a 1 that could be used for immunotherapy purposes in future.

Peptidase Activity of the Escherichia coli Hsp31 Chaperone

Hsp31, the Escherichia coli hcha gene product, is a molecular chaperone whose activity is inhibited by ATP at high temperature. Its crystal structure reveals a putative Cys(184), His(185), and Asp(213) catalytic triad similar to that of the Pyrococcus horikoshii protease PH1704, suggesting that it should display a proteolytic activity. A preliminary report has shown that Hsp31 has an exceedingly weak proteolytic activity toward bovine serum albumin and a peptidase activity toward two peptide substrates with small amino acids at their N terminus (alanine or glycine), but the physiological significance of this observation remains unclear. In this study, we report that Hsp31 does not diplay any significant proteolytic activity but has peptidolytic activity. The aminopeptidase cleavage preference of Hsp31 is Ala > Lys > Arg > His, suggesting that Hsp31 is an aminopeptidase of broad specificity. Its aminopeptidase activity is inhibited by the thiol reagent iodoacetamide and is completely abolished in a C185A mutant, which is consistent with Hsp31 being a cysteine peptidase. The aminopeptidase activity of Hsp31 is also inhibited by EDTA and 1,10-phenanthroline, in concordance with the importance of the putative His(85), His(122), and Glu(90) metal-binding site revealed by crystallographic studies. An Hsp31-deficient mutant accumulates more 8-12-mer peptides than its parental strain, and purified Hsp31 can transform these peptides into smaller peptides, suggesting that Hsp31 has an important peptidase function both in vivo and in vitro. Proteins interacting with Hsp31 have been identified by reverse purification of a crude E. coli extract on an Hsp31-affinity column, followed by SDS-polyacrylamide electrophoresis and mass spectrometry. The ClpA component of the ClpAP protease, the chaperone GroEL, elongation factor EF-Tu, and tryptophanase were all found to interact with Hsp31, thus substantiating the role of Hsp31 as both chaperone and peptidase.

Further isolation and characterization of grammistins from the skin secretion of the soapfish Grammistes sexlineatus

Soapfishes contain peptide toxins (grammistins) in the skin secretion. Two grammistins (Gs 1 and Gs 2) and six grammistins (Pp 1, Pp 2a, Pp 2b, Pp 3, Pp 4a and Pp 4b) have already been isolated from Grammistes sexlineatus and Pogonoperca punctata, respectively. In this study, five grammistins (Gs A–E), together with grammistins Gs 1 and Gs 2, were further isolated from G. sexlineatus by gel filtration and reverse-phase HPLC. Sequence analyses revealed that grammistins Gs A (28 residues) and Gs C (26 residues) are analogous to grammistin Pp 3 and grammistin Gs B (12 residues) to grammistin Pp 1, while grammistins Gs D (13 residues) and Gs E (13 residues) are identical with grammistins Pp 1 and Pp 2b, respectively. Grammistins Gs A–C exhibited antibacterial activity with a broad spectrum against nine species of bacteria in common with the other grammistins but had no hemolytic activity differing from the other grammistins. Grammistins Gs A–E, Gs 1 and Gs 2 could release carboxyfluorescein entrapped within liposomes made of either phosphatidylcholine or phosphatidylglycerol/phosphatidylcholine (3:1), demonstrating their membrane-lytic activity. However, no clear relationship between the membrane-lytic activity and the biological activity of grammistins was recognized.

Purification and biochemical characterization of a thermostable, alkaliphilic, extracellular alpha-amylase from Bacillus subtilis DM-03, a strain isolated from the traditional fermented food of India.

Bacillus subtilis strain DM-03, which is isolated from starter culture used for the production of alcohol by local Assam tribes, grows optimally at 52-55 degrees C and secretes a significant amount of alpha-amylase at pH 8.0 into the culture media. This alpha-amylase, purified by ion-exchange, gel-filtration and reverse-phase HPLC, constitutes 2.9% of the total extracellular protein. This purified enzyme, named Bsamy-I, has a subunit with molecular mass of 42.8 kDa as determined by SDS/PAGE, and optimum temperature and pH values at 52-55 degrees C and 9.0 respectively, which makes it ideal for use in the detergent industries. Maximum alpha-amylase production is obtained by using soluble starch and NH(4)Cl as carbon and nitrogen sources respectively. Thermostability of the enzyme is evident from heating the enzyme at 95 degrees C for 10 min, which results in a loss of 60% of the original enzyme activity. 4-Bromophenacyl bromide and PMSF at 4 and 1.5 mM concentration respectively completely abolish the enzymic activity, documenting the essential role of histidine and carboxylic residues in the catalytic process.

Allergenicity of crustacean extractives and its reduction by protease digestion

This study was undertaken to evaluate and reduce the allergenicity of crustacean extractives that are added to a variety of processed foods as seasonings. Tropomyosin, the major crustacean allergen, was detected in only one of the six kinds of crustacean extractives by SDS–PAGE and immunoblotting. However, all the extractives were shown to be considerably allergenic by inhibition ELISA using sera from crustacean-allergic patients. Analyses by gel filtration HPLC suggested that tropomyosin is mostly degraded to peptide fragments during manufacturing of the extractives but at least some of the fragments are still IgE-reactive. On digestion with proteases, the allergenicity of the extractives was almost completely lost. The effectiveness of protease digestion to reduce the allergenicity of tropomyosin was also confirmed in model experiments using the heated extracts from four species of crustaceans.

Speciation of selenocompounds by capillary HPLC coupled with ICP-MS using multi-mode gel filtration columns

Multi-mode gel filtration HPLC columns of capillary size coupled with ICP-MS were evaluated as to the separation of naturally occurring selenocompounds. The major selenocompounds found in selenized garlic, i.e., Se-methylselenocysteine and γ-glutamyl-Se-methylselenocysteine, were separated on the narrower column (GS320A-M5D, 0.5 mm i.d. × 150 mm). The selenometabolites found in the urine of a selenium-toxicosis rat, i.e., selenosugar (1-β-methylseleno-N-acetyl-D-galactosamine) and trimethylselenonium, were also separated on the same column. On the other hand, the selenoamino acids, i.e., Se-methylselenocysteine and selenomethionine, were separated on the larger column (GS320A-M8E, 0.8 mm i.d. × 250 mm). These samples were introduced directly into the capillary columns without any pretreatment. The capillary HPLC separated these selenocompounds into distinct peaks with satisfactory sensitivity and a better S/N ratio than conventional HPLC despite that the sample volume was reduced to 1/200 (100 nl).

αA-Crystallin Interacting Regions in the Small Heat Shock Protein, αB-Crystallin

Amino acid sequences of alphaB-crystallin, involved in interaction with alphaA-crystallin, were determined by using peptide scans. Positionally addressable 20-mer overlapping peptides, representing the entire sequence of alphaB-crystallin, were synthesized on a PVDF membrane. The membrane was blocked with albumin and incubated with purified alphaA-crystallin. Probing the membrane with alphaA-crystallin-specific antibodies revealed residues 42-57, 60-71, and 88-123 in alphaB-crystallin to interact with alphaA-crystallin. Residues 42-57 and 60-71 interacted more strongly with alphaA-crystallin than the 88-123 sequence of alphaB-crystallin. Binding of one of the alphaB peptides (42-57) to alphaA-crystallin was also confirmed by gel filtration studies and HPLC analysis. The alphaB-crystallin sequences involved in interaction with alphaA-crystallin were distinct from the chaperone sites reported earlier as binding of the alphaB sequence from residues 42-57 does not alter the chaperone-like function of alphaA-crystallin. To identify the critical residues involved in interaction with alphaA-crystallin, R50G and P51A mutants of alphaB-crystallin were made and tested for their ability to interact with alphaA-crystallin. The oligomeric size and hydrophobicity of the mutants were similar. Circular dichroism studies showed that the P51A mutation increased the alpha-helical content of the protein. While the alphaBR50G mutant showed chaperone-like activity similar to wild-type alphaB, alphaBP51A showed reduced chaperone function. Fluorescence resonance energy transfer studies showed that the P51A mutation decreased the rate of subunit exchange with alphaA by 63%, whereas the R50G mutation reduced the exchange rate by 23%. Similar to wild-type alphaB, alphaB-crystallin peptide (42-57) effectively competed with alphaBP51A and alphaBR50G for interaction with alphaA. Thus, our studies showed that the alphaB-crystallin sequence (42-57) is one of the interacting regions in alphaB and alphaA oligomer formation.

Isolation and molecular cloning of novel peptide toxins from the sea anemone Antheopsis maculata

Three peptide toxins (Am I–III) with crab toxicity were isolated from the sea anemone Anthopleura maculata by gel filtration and reverse-phase HPLC. Am I was weakly lethal to crabs (LD 50 830 μg/kg) and Am III was potently lethal (LD 50 70 μg/kg), while Am II was only paralytic (ED 50 420 μg/kg). The complete amino acid sequences of the three toxins were determined by cDNA cloning based on 3′-Race and 5′-Race. Although Am III (47 residues) is an analogue of the well-known type 1 sea anemone sodium channel toxins, both Am I (27 residues) and II (46 residues) are structurally novel peptide toxins. Am I is a new toxin having no sequence homologies with any toxins. Am II shares 28–39% identity with the recently characterized sea anemone toxins inhibiting specialized ion channels, BDS-I and II from Anemonia sulcata and APETx1 and 2 from Anthopleura elegantissima. The precursor proteins of the three toxins are commonly composed of a signal peptide, a propart with a pair of basic residues (Lys-Arg) at the end and the remaining portion. Very interestingly, the Am I precursor protein contains as many as six copies of Am I.

Isolation and cDNA Cloning of Ovarian Cortical Rod Protein in Kuruma Prawn Marsupenaeus japonicus (Crustacea: Decapoda: Penaeidae)

Two cortical rod proteins having molecular weights of 28.6 kDa and 30.5 kDa were isolated from the mature ovary of Marsupenaeus japonicus using gel filtration and reversed-phase HPLC. Analysis of the N-terminal amino acid sequence of the 28.6 kDa molecule revealed that amino acid residues 1-21 corresponded to residues 9-29 of the 30.5 kDa molecule. Examination of homology using BLAST showed that 21 amino acids out of 29 residues of the 28.6 kDa molecule, and 14 out of 29 residues of the 30.5 kDa molecule were identical to that of the ovarian cortical rod proteins of Penaeus semisulcatus. Positive immunohistochemical reaction to antiserum raised against the 28.6 kDa protein was observed on cortical rods forming around the periphery of oocytes at the maturation stages. Western blotting analysis revealed that both the 28.6 kDa and 30.5 kDa molecules stained with the anti-28.6 kDa antiserum. Furthermore, the 28.6 kDa and 30.5 kDa proteins were both glycosylated, as evidenced by positive carbohydrate staining using Concanavalin A and production of positive PAS reaction. These results indicate that the cortical rods are comprised of the 28.6 kDa and 30.5 kDa molecules. We subsequently cloned two full-length cDNAs based on the N-terminal sequences of the 28.6 kDa and 30.5 kDa molecules. The open reading frame of 28.6 kDa and 30.5 kDa encoded 276 amino acid residues. Comparison analysis of the two cDNAs revealed that the location of the processing site and sequence of signal peptides differed, indicating that the two cDNAs are products of two separate genes and encode the 28.6 kDa molecule and 30.5 kDa molecule, respectively. Both proteins possessed one potential N-linked glycosylation site. It is considered that both molecules are components of the cortical rods, forming a jelly layer after fertilization.

Anthocyanins from fruits of Rubus pinnatus and Rubus rigidus

The same anthocyanins, cyanidin 3-(6″- O-α-rhamnopyranosyl-β-glucopyranoside) ( 1), and cyanidin-3- O-β-glucopyranoside ( 2), were isolated from extracts of red fruits of Rubus pinnatus Willd. and purple-black fruits of Rubus rigidus Sm. using Amberlite XAD-7 column chromatography, Sephadex LH-20 gel filtration and preparative HPLC. Their structures were elucidated by a combination of chromatography, homo- and heteronuclear NMR techniques. The relative amounts of 1 and 2 in R. rigidus were 59.4% and 40.6%, respectively, while in R. pinnatus, the relative amounts were 58.6% and 41.4%, respectively.

A Serine Protease from a Detergent-soluble Extract of Leishmania (Leishmania) amazonensis

Proteases mediate important crucial functions in parasitic diseases, and their characterization contributes to the understanding of host-parasite interaction. A serine protease was purified about 43-fold with a total recovery of 60% from a detergent-soluble extract of promastigotes of Leishmania amazonensis. The purification procedures included aprotinin-agarose affinity chromatography and gel filtration high performance liquid chromatography. The molecular mass of active enzyme was 110 kDa by native gel filtration HPLC and by SDS-PAGE gelatin under non-reducing conditions. Under conditions of reduction using SDS-PAGE gelatin analyses the activity of enzyme was observed in two proteins of 60 and 45 kDa, suggesting that the enzyme may be considered as a dimer. The Leishmania protease was not glycosylated, and its isoelectric point (pI) was around 4.8. The maximal protease activity was at pH 7.0 and 28 degrees C, using a-N-o-tosyl-L-arginyl-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that this enzyme was totally denatured after pre-treatment at 42 degrees C for 12 min and preserved only 20% of its activity after pre-treatment at 37 degrees C for 24 h, in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin and gelatin were hydrolyzed by Leishmania protease. Inhibition studies indicated that the enzyme belonged to a serine protease class because of a significant impediment by serine protease inhibitors such as benzamidine, aprotinin, and antipain. The activity of the present serine protease is negatively modulated by calcium and zinc and positively modulated by manganese ions. This is the first study that reports the purification of a protease from a detergent-soluble extract of Leishmania species.

Purification and characterization of an acid deoxyribonuclease from the cultured mycelia of Cordyceps sinensis.

A new acid deoxyribonuclease (DNase) was purified from the cultured mycelia of Cordyceps sinensis, and designated CSDNase. CSDNase was purified by (NH(4))(2)SO(4) precipitation, Sephacryl S-100 HR gel filtration, weak anion-exchange HPLC, and gel filtration HPLC. The protein was single-chained, with an apparent molecular mass of ca. 34 kDa, as revealed by SDS-PAGE, and an isoelectric point of 7.05, as estimated by isoelectric focusing. CSDNase acted on both double-stranded (ds) and single- stranded (ss) DNA, but preferentially on dsDNA. The optimum pH of CSDNase was pH 5.5 and its optimum temperature 55. The activity of CSDNase was not dependent on divalent cations, but its enzymic activity was inhibited by high concentration of the cation: MgCl(2) above 150 mM, MnCl(2) above 200 mM, ZnCl(2) above 150 mM, CaCl(2) above 200 mM, NaCl above 300 mM, and KCl above 300 mM. CSDNase was found to hydrolyze DNA, and to generate 3-phosphate and 5-OH termini. These results indicate that the nucleolytic properties of CSDNase are essentially the same as those of other well-characterized acid DNases, and that CSDNase is a member of the acid DNase family. To our knowledge, this is the first report of an acid DNase in a fungus.

Cloning and characterization of a gene encoding the gamma-butyrolactone autoregulator receptor from Streptomyces clavuligerus.

With primers designed for the conserved region of the gamma-butyrolactone autoregulator receptor proteins from Streptomyces species, PCR using the Streptomyces clavuligerus genome DNA as a template gave a clear band of 100 bp, the sequence of which revealed high similarity to the expected region of a receptor gene. By Southern blot and colony hybridization with the 100-bp insert as a probe, plasmid pSCA, harboring a 4.2 kb- SalI fragment, was obtained. Sequence analysis on the insert revealed a 702-bp ORF encoding a protein with a moderate similarity (identity, 33-43%; similarity, 51-62%) to known gamma-butyrolactone autoregulator receptor proteins from Streptomyces sp. The ORF was named scaR ( S. clavuligerus autoregulator receptor). The scaR/pET-3d plasmid was constructed for overexpression of the recombinant ScaR protein (rScaR) in Escherichia coli, and the rScaR protein was purified to homogeneity by DEAE-ion-exchange HPLC. The molecular mass of the purified rScaR protein was determined to be 27 kDa as determined by SDS-PAGE, and 54 kDa by gel filtration HPLC under nondenatured conditions at a low protein concentration, indicating that the majority of the native ScaR is present in the form of a dimer, although rScaR tended to aggregate into a higher molecular form of 230 kDa at a high protein concentration. A binding assay with tritium-labeled autoregulators indicated that IM-2 type compounds with a long C2 side chain were the most effective ligands for rScaR, demonstrating for the first time that the beta-lactam producer S. clavuligerus contains a gene for the gamma-butyrolactone autoregulator receptor.

Prebiotic Inhibitory Activity of Protein-Like Molecules to the Template-Directed Formation of Oligoguanylate from Guanosine 5′-Monophosphate 2-Methylimidazolide on a Polycytidylic Acid Template

Abstract The influence of thermal copolymers of amino acids (TCAA) as a primitive protein enzyme model has been investigated to detect catalytic and/or inhibitory activities on the template-directed formation of oligoguanylate (oligo(G)) from guanosine 5′-monophosphate 2-methylimidazolide (2-MeImpG) on a polycytidylic acid template (poly(C)) (TD reaction). Different amino acids and compositions were used to prepare TCAAs. These TCAAs were characterized by gel filtration HPLC and the composition analysis of amino acids using phenyl isothiocyanate. It was found that TCAA including His (TCAA-His) has inhibitory activity to the TD reaction, in which TCAA-His accelerates the hydrolysis of 2-MeImpG. Other types of TCAAs, including Gly, Ala, Val, Glu, and Asp, also accelerate the formation of P1,P2-bis(5′-guanosyl) diphosphate (G5′ppG) and G5′ppG incorporated oligo(G)s during the TD reaction at high concentrations of TCAAs. These reactions are considered as primitive enzyme models for the hydrolysis of nucleotide monomer and the formation of pyrophosphate-containing oligo(G).

Leishmania (Leishmania) amazonensis: purification and characterization of a promastigote serine protease

Pathogenic protozoan proteases play crucial roles in the host–parasite interaction, and its characterization contributes to the understanding of protozoan disease mechanisms. A Leishmania amazonensis promastigote protease was purified 36-fold, using aprotinin–agarose affinity chromatography and gel filtration high performance liquid chromatography, yielding a total recovery of 49%. The molecular mass of active enzyme obtained from native gel filtration HPLC and SDS–PAGE under conditions of reduction and non-reduction was 68 kDa, suggesting that the enzyme may exist as a monomer. The protease isoelectric point (p I) was around 4.45 and, as demonstrated by deglycosylation assay, it did not have any carbohydrate content. The optimal pH and temperature of the enzyme were 8.0 and 28 °C, respectively, determined using α- N-ρ-tosyl- l-arginyl-methyl ester ( l-TAME) as substrate. Assays of thermal stability indicated that 50% of the enzymatic activity was preserved after 4 min of pre-treatment at 42 °C and after 24 h of pre-treatment at 37 °C, both in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin, and both gelatin and peptide substrates containing arginine in ester bound were hydrolyzed by 68 kDa protease. The insulin β-chain was also hydrolyzed by the protease, and four peptidic bonds (L 11–V 12, E 13–A 14, L 15–Y 16, and Y 16–L 17) were susceptible to the 68-kDa protease action. Inhibition studies suggested that the enzyme belonged to a serine protease class inhibited by calcium ions and activated by manganese ions. These findings demonstrate that the L. amazonensis 68-kDa serine protease differs from those of other protozoan parasites.

Expression, purification, and characterization of a novel methyl parathion hydrolase

The mpd gene coding for a novel methyl parathion hydrolase (MPH) was previously reported and its putative open reading frame was also identified. To further confirm its coding region, the intact region encoding MPH was obtained by PCR and expressed in Escherichia coli as a hexa-His C-terminal fusion protein. The fusion protein was purified to homogeneity by metal-affinity chromatography. The enzyme activity and zymogram assay showed that the fusion protein was functional in degrading methyl parathion. The amino terminal sequencing of the purified recombinant MPH indicated that a signal peptide of the first 35 amino acids was cleaved from its precursor to form active MPH. A rat polyclonal antiserum was raised against the purified mature fusion protein. The results of Western blot and zymogram demonstrated that mature MPH in native Plesiomonas sp. strain M6 was also processed from its precursor by cleavage of a putative signal peptide at the amino terminus. The production of active MPH in E. coli was greatly improved after the coding region for the signal peptide was deleted. HPLC gel filtration of the purified mature recombinant MPH revealed that the MPH was a monomer.

Saliva of the Yellow Fever mosquito, Aedes aegypti, modulates murine lymphocyte function

Saliva of many vector arthropods contains factors that inhibit haemostatic responses in their vertebrate hosts. Less is known about the effect of vector saliva on host immune responses. We investigated the effect of Aedes aegypti salivary gland extracts on antigen-stimulated responses of transgenic OVA-TCR DO11 mouse splenocytes in vitro. T-cell proliferation was inhibited in a dose-dependent manner, with greater than 50% inhibition at 0.3 salivary gland pair (SGP) equivalents/mL. LPS-stimulated B-cell proliferation was also inhibited. Secretion of the Th1 cytokines IL-2 and IFN-gamma was reduced by 50% or more with 0.45-0.6 SGP/mL, as was secretion of the pro-inflammatory cytokines GM-CSF and TNF-alpha, and the Th2 cytokine IL-5. The Th2 cytokines IL-4 and IL-10 were similarly reduced with 0.6-2 SGP/mL. Inhibition of lymphocyte function involved modulation of viable T-cells at low salivary gland extract (SGE) concentrations, and decreased viability at higher concentrations. Dendritic cells were not killed by salivary gland extracts at concentrations as high as 25 salivary gland pairs/mL, but secretion of IL-12 was inhibited by 87% following exposure to 0.6 SGP/mL. Activity is present in saliva and extracts of female but not male salivary glands, and it is depleted from salivary glands of blood-fed mosquitoes. The activity is denatured by boiling and by digestion with the protease papain, indicating a protein; gel filtration HPLC indicates a mass of about 387 kDa. These results suggest that A. aegypti saliva exerts a marked immunomodulatory influence on the environment at the bite site.

Purification and properties of proteinaceous trypsin inhibitors in the skin mucus of pufferfish Takifugu pardalis

A screening assay for inhibitory activity against trypsin in skin mucus from 29 species of fishes reveals a wide distribution of trypsin inhibitors in skin mucus and relatively high antitryptic activity in pufferfish of the family Tetraodontidae. Two trypsin inhibitors termed TPTI 1 and 2 were purified to homogeneity from the skin mucus of Takifugu pardalis by salting out, lectin affinity, anion exchange FPLC and gel filtration HPLC. Both inhibitors are acidic glycoproteins, with an apparent molecular mass of 57 kDa in SDS-PAGE, p I below 4 and 1.9% reducing sugar for TPTI 1 and with an apparent molecular mass of 47 kDa in SDS-PAGE, p I 5.2 and 0.8% reducing sugar for TPTI 2. The inhibitors effectively repress the catalytic activity of trypsin and α-chymotrypsin, and therefore can be classified as serine protease inhibitors. The inhibitory constants against trypsin were 4.9×10 −8 M for TPTI 1 and 3.9×10 −8 M for TPTI 2. Both inhibitors react with trypsin at a molar ratio of 1:1, although TPTI 1 reversibly inactivates the proteolytic activity of trypsin non-competitively and TPTI 2, competitively. The trypsin inhibitors in the skin mucus of T. pardalis may function as defense substances to neutralize serine proteases released by invasive pathogens.

Proton dependence of tobacco mosaic virus dissociation by pressure

Tobacco mosaic virus (TMV) is an intensely studied model of viruses. This paper reports an investigation into the dissociation of TMV by pH and pressure up to 220 MPa. The viral solution (0.25 mg/ml) incubated at 277 K showed a significant decrease in light scattering with increasing pH, suggesting dissociation. This observation was confirmed by HPLC gel filtration and electron microscopy. The calculated volume change of dissociation (Δ V) decreased (absolute value) from −49.7 ml/mol of subunit at pH 3.8 to −21.7 ml/mol of subunit at pH 9.0. The decrease from pH 9.0 to 3.8 caused a stabilization of 14.1 kJ/mol of TMV subunit. The estimated proton release calculated from pressure-induced dissociation curves was 0.584 mol H +/mol of TMV subunit. These results suggest that the degree of virus inactivation by pressure and the immunogenicity of the inactivated structures can be optimized by modulating the surrounding pH.