Showing papers on "Proteolytic enzymes published in 1991"

Inflammation in the course of early myocardial ischemia.

Abstract: Experimental models of acute ischemic myocardial injury indicate that the inflammatory response after the ischemic event contributes to tissue damage. This is especially apparent with reperfusion of the ischemic tissue. In such models some therapeutic strategies designed to reduce neutrophil accumulation or function have resulted in apparently beneficial effects. Although such findings are encouraging, interventions into these pathological processes using specific molecular targets will require greater understanding of specific mechanisms. Current evidence indicates that potential sites of therapeutic intervention will be found in pathways leading to complement activation, generation of lipid-derived mediators, adhesion of neutrophils to endothelial cells and cardiac myocytes, and activation of neutrophil secretory processes releasing, for example, proteolytic enzymes and reactive oxygen. Understanding the dynamic interplay between the mediators, adhesion pathways, and secretory processes that results in myocardial damage will allow a rational approach to controlling the detrimental inflammatory consequences of ischemia and reperfusion.

Inhibition of food-borne bacterial pathogens by bacteriocins from lactic acid bacteria isolated from meat.

Abstract: Ten strains of bacteriocin-producing lactic acid bacteria were isolated from retail cuts of meat. These 10 strains along with 11 other bacteriocin-producing lactic acid bacteria were tested for inhibitory activity against psychotrophic pathogens, including four strains of Listeria monocytogenes, two strains of Aeromonas hydrophila, and two strains of Staphylococcus aureus. Inhibition due to acid, hydrogen peroxide, and lytic bacteriophage were excluded. The proteinaceous nature of the inhibitory substance was confirmed by demonstration of its sensitivity to proteolytic enzymes. Eight of the meat isolates had inhibitory activity against all four L. monocytogenes strains. Bacteriocin activity against L. monocytogenes was found in all of the strains obtained from other sources. Activity against A. hydrophila and S. aureus was also common.

Characterization of leucocin A-UAL 187 and cloning of the bacteriocin gene from Leuconostoc gelidum.

Abstract: Leucocin A-UAL 187 is a bacteriocin produced by Leuconostoc gelidum UAL 187, a lactic acid bacterium isolated from vacuum-packaged meat. The bacteriocin was purified by ammonium sulfate or acid (pH 2.5) precipitation, hydrophobic interaction chromatography, gel filtration, and reversed-phase high-performance liquid chromatography with a yield of 58% of the original activity. Leucocin A is stable at low pH and heat resistant, and the activity of the pure form is enhanced by the addition of bovine serum albumin. It is inactivated by a range of proteolytic enzymes. The molecular weight was determined by mass spectrometry to be 3,930.3 +/- 0.4. Leucocin A-UAL 187 contains 37 amino acids with a calculated molecular weight of 3,932.3. A mixed oligonucleotide (24-mer) homologous to the sequence of the already known N terminus of the bacteriocin hybridized to a 2.9-kb HpaII fragment of a 7.6-MDa plasmid from the producer strain. The fragment was cloned into pUC118 and then subcloned into a lactococcal shuttle vector, pNZ19. DNA sequencing revealed an operon consisting of a putative upstream promoter, a downstream terminator, and two open reading frames flanked by a putative upstream promoter and a downstream terminator. The first open reading frame downstream of the promoter contains 61 amino acids and is identified as the leucocin structural gene, consisting of a 37-amino-acid bacteriocin and a 24-residue N-terminal extension. No phenotypic expression of the bacteriocin was evident in several lactic acid bacteria that were electrotransformed with pNZ19 containing the 2.9-kb cloned fragment of the leucocin A plasmid.

Factors in virulence expression and their role in periodontal disease pathogenesis.

Abstract: The classic progression of the development of periodontitis with its associated formation of an inflammatory lesion is characterized by a highly reproducible microbiological progression of a Gram-positive microbiota to a highly pathogenic Gram-negative one. While this Gram-negative microbiota is estimated to consist of at least 300 different microbial species, it appears to consist of a very limited number of microbial species that are involved in the destruction of periodontal diseases. Among these "putative periodontopathic species" are members of the genera Porphyromonas, Bacteroides, Fusobacterium, Wolinella, Actinobacillus, Capnocytophaga, and Eikenella. While members of the genera Actinomyces and Streptococcus may not be directly involved in the microbial progression, these species do appear to be essential to the construction of the network of microbial species that comprise both the subgingival plaque matrix. The temporal fluctuation (emergence/disappearance) of members of this microbiota from the developing lesion appears to depend upon the physical interaction of the periodontal pocket inhabitants, as well as the utilization of the metabolic end-products of the respective species intimately involved in the disease progression. A concerted action of the end-products of prokaryotic metabolism and the destruction of host tissues through the action of a large number of excreted proteolytic enzymes from several of these periodontopathogens contribute directly to the periodontal disease process.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased expression of the Mr 72,000 type IV collagenase in human colonic adenocarcinoma.

Abstract: Proteolytic enzymes, such as type IV collagenase, play an important role in tumor invasion and metastasis. To examine Mr 72,000 type IV collagenase expression in human colon carcinoma, blot hybridization of total RNA from 19 primary colon tumors were performed. These filters were probed with complementary DNA probes encoding the Mr 72,000 type IV collagenase metalloenzyme. The results were expressed as the ratio of the messenger RNA (mRNA) levels in the tumor tissue to that in the adjacent normal mucosa (R). The level of the 3.1-kilobase type IV collagenase mRNA was higher in the primary tumor than in the normal adjacent colonic mucosa in 13 of 18 (72%) cases with a diagnosis of adenocarcinoma. These cases were divided into high expression (R, 4.50 to 29.34) and intermediate expression (R, 2.54 to 3.31) subgroups. Both groups showed statistically significant (P less than 0.05) elevations when compared with the five cases showing the lowest levels of Mr 72,000 type IV collagenase mRNA expression (low expression subgroup; R, 0.96 to 1.48). With this demonstrated elevation of Mr 72,000 type IV collagenase mRNA in colorectal adenocarcinoma we examined concomitant expression at the protein level using immunohistochemical techniques. Immunohistochemical examination of 70 cases of colon tumors, including 30 benign adenomas, using anti-Mr 72,000 type IV collagenase antibodies demonstrated a significant correlation with Duke's classification (P less than 0.001). Our results suggest that enhanced expression of the Mr 72,000 type IV collagenase enzyme may be a marker of human colorectal tumor invasiveness.

Determination of the intracellular sites and topology of glucosylceramide synthesis in rat liver

Abstract: We examined the intracellular site(s) and topology of glucosylceramide (GlcCer) synthesis in subcellular fractions from rat liver, using radioactive and fluorescent ceramide analogues as precursors, and compared these results with those obtained in our recent study of sphingomyelin (SM) synthesis in rat liver [Futerman, Stieger, Hubbard & Pagano (1990) J. Biol. Chem. 265, 8650-8657]. In contrast with SM synthesis, which occurs principally at the cis/medial Golgi apparatus, GlcCer synthesis was more widely distributed, with substantial amounts of synthesis detected in a heavy (cis/medial) Golgi-apparatus subfraction, a light smooth-vesicle fraction that is almost devoid of an endoplasmic-reticulum marker enzyme (glucose-6-phosphatase), and a heavy vesicle fraction. Furthermore, no GlcCer synthesis was detected in an enriched plasma-membrane fraction after accounting for contamination by Golgi-apparatus membranes. These results suggest that a significant amount of GlcCer may be synthesized in a pre- or early Golgi-apparatus compartment. Unlike SM synthesis, which occurs at the luminal surface of the Golgi apparatus, GlcCer synthesis appeared to occur at the cytosolic surface of intracellular membranes, since (i) limited proteolytic digestion of intact Golgi-apparatus vesicles almost completely inhibited GlcCer synthesis, and (ii) the extent of UDP-glucose translocation into the Golgi apparatus was insufficient to account for the amount of GlcCer synthesis measured. These findings imply that, after its synthesis, GlcCer must undergo transbilayer movement to the luminal surface to account for the known topology of higher-order glycosphingolipids within the Golgi apparatus and plasma membrane.

The induction of a specific protease for insulin-like growth factor binding protein-3 in the circulation during severe illness.

Abstract: The insulin-like growth factors (IGF-I and IGF-II) are almost completely bound in the circulation to specific binding proteins (IGFBPs). These IGFBPs appear to play a pivotal role in maintaining circulating levels and modulating the delivery of the IGFs to the tissues. A large proportion of the circulating IGFs are bound with high affinity to one of the binding proteins. IGFBP-3. The mechanism by which these IGFs are transferred from the circulatory pool to the tissue receptors is at present unclear. Recent studies in late pregnancy have demonstrated the presence of specific proteases which may modify the IGFBPs such that their affinities for the IGFs are reduced. In this paper, we have demonstrated the presence of a heat-sensitive cation-dependent proteolytic enzyme specific for IGFBP-3 in the serum of five severely ill patients. The activity of this protease was found to vary in these patients, becoming more apparent during fasting than when studied after commencement of parenteral nutrition, indicating that one of the influencing factors in the activity of this protease is the nutritional intake of the patient. Age- and sex-matched healthy adults were also studied in a similar protocol, but no proteolytic modification of any of the IGFBPs was found in any of the samples examined. As the levels of both IGF-I and IGF-II were found to be low in the patients, the presence of a circulatory protease suggests that this may be an adaptive response to increase the bioavailability of the IGFs and possibly to improve the nitrogen retention and counter the catabolic state in severe illness.

A parametric study ot protease production in batch and fed‐batch cultures of Bacillus firmus

Abstract: Proteolytic enzymes produced by Bacillus species find a wide variety of applications in brewing, detergent, food, and leather industries. Owing to significant differences normally observed in culture conditions promoting cell growth and those promoting production of metabolites such as enzymes, for increased efficacy of bioreactor operations it is essential to identify these sets of conditions (including medium formulation). This study is focused on formulation of a semidefined medium that substantially enhances synthesis and secretion of an alkaline protease in batch cultures of Bacillus firmus NRS 783, a known superior producer of this enzyme. The series of experiments conducted to identify culture conditions that lead to improved protease production also enables investigation of the regulatory effects of important culture parameters including pH, dissolved oxygen, and concentrations of nitrogen and phosphorous sources and yeast extract in the medium on cell growth, synthesis and secretion of protease, and production of two major nonbiomass products, viz., acetic acid and ethanol. Cell growth and formation of the three nonbiomass products are hampered significantly under nitrogen, phosphorous, or oxygen limitation, with the cells being unable to grow in an oxygen-free environment. Improvement in protease production is achieved with respect to each culture parameter, leading in the process to 80% enhancement in protease activity over that attained using media reported in the literature. Results of a few fed-batch experiments with constant feed rate, conducted to examine possible enhancement in protease production and to further investigate repression of protease synthesis by excess of the principal carbon and nitrogen sources, are also discussed. The detailed investigation of stimulatory and repressory effects of simple and complex nutrients on protease production and metabolism of Bacillus firmus conducted in this study will provide useful guidelines for design of bioreactors for production of protease and bulk chemicals by this bacterium.

The core element of the EcoRII methylase as defined by protease digestion and deletion analysis.

Abstract: Binding of the EcoRII DNA methyltransferase to azacytosine-containing DNA protects the enzyme from digestion by proteases. The limit digest yields a product having a Mr on SDS-PAGE 20% less than the intact protein. The N terminus of the tryptic digestion product was sequenced and found to be missing the N terminal 82 amino acids. Under the conditions used unbound enzyme was digested to small peptides. Protection of the enzyme from protease digestion implies that the enzyme undergoes major conformational changes when bound to DNA. The trypsin sensitive region of the EcoRII methyltransferase occurs prior to the first constant region shared with other procaryotic DNA(cytosine-5)methyltransferases. To determine if this region played a role in substrate binding or specificity, N-terminal deletion mutants were studied. Deletion of 97 amino acids resulted in a decrease of enzyme activity. Further deletions caused a complete loss of activity. Enzyme deleted through amino acid 85 was purified and found to have the same specificity as wild type however there was an increase in Km for both S-adenosylmethionine (AdoMet) and DNA of 27 and 18 fold respectively. The N-terminus of the EcoRII methylase, although a variable region present in many procaryotic DNA(cytosine-5)methylases, plays no role in determining enzyme specificity, although it does contribute to the interaction with both AdoMet and DNA.

RGD-dependent entry of coxsackievirus A9 into host cells and its bypass after cleavage of VP1 protein by intestinal proteases.

Abstract: The recently reported nucleotide sequence of coxsackievirus A9 (CAV-9) showed that unlike other enteroviruses, CAV-9 has an insertion of about 17 amino acids at the C-terminal end of VP1 (K. H. Chang, P. Auvinen, T. Hyypia, and G. Stanway, J. Gen. Virol. 70:3269-3280, 1989). This sequence includes the RGD (arginine-glycine-aspartic acid) motif which is known to be important in certain protein-protein interactions. We studied the inhibitory effect of RGD-containing peptides in the attachment of CAV-9 to African green monkey kidney cells. A peptide corresponding to the RRGDM sequence derived from the inserted segment of CAV-9 was found to block virus attachment effectively, and the inhibition was dose dependent. Substitution of glutamic acid for the homologous aspartic acid completely abolished the inhibitory effect, indicating great specificity of the action. During replication in the gut, all enteroviruses are exposed to host proteolytic enzymes. Exposure of CAV-9 to purified trypsin or human intestinal fluid resulted in selective cleavage of the VP1 capsid protein. Intact and trypsin-cleaved VP1 proteins gave identical N-terminal sequences, indicating that cleavage of VP1 takes place near the C terminus. Attachment of proteolytically cleaved infectious CAV-9 to green monkey kidney cells was not prevented by RGD-containing peptides, indicating that cleaved CAV-9 is able to bypass RGD-dependent entry. The altered receptor specificity of proteolytically cleaved viruses may have important consequences in the pathogenesis of enteric infections. Images

Lipoproteins of Borrelia burgdorferi and Treponema pallidum activate cachectin/tumor necrosis factor synthesis. Analysis using a CAT reporter construct.

Abstract: Lipoproteins from two pathogenic spirochetes (Borrelia burgdorferi and Treponema pallidum) induced the biosynthesis of TNF in murine macrophages and in permanently transformed macrophages of the cell line RAW 264.7. Induction was studied by measuring the secretion of biologically active TNF and by measuring the activity of the reporter enzyme chloramphenicol acetyltransferase (CAT) produced within macrophages transfected with an endotoxin-responsive CAT construct. Several lines of evidence indicated that the induction of TNF and CAT was attributable to the spirochete lipoproteins rather than to contaminating or endogenous LPS: 1) the dose response curves observed for the lipoproteins were markedly different from those obtained with LPS; 2) lipoprotein-mediated activation was unaffected by amounts of polymyxin B that completely neutralized the induction of TNF and CAT by LPS, 3) low concentrations of the lipoproteins induced TNF in macrophages from endotoxin-unresponsive C3H/HeJ mice as effectively as in macrophages from normal C3H/HeN mice, and 4) isolated spirochete lipoproteins, but not a non-lipoprotein immunogen, were potent inducers of CAT in the transformed macrophages. Moreover, LPS was not detected in the B. burgdorferi lipoprotein mixtures by Limulus amebocyte lysate assay. Proteolytic digestion of the intact bacterial protein preparations only modestly diminished their ability to activate the cells, suggesting that small lipopeptides comprise the biologically active portions of the molecules, as is the case with the murein lipoprotein of Escherichia coli. Through their ability to induce TNF production by macrophages, spirochete lipoproteins may play important roles in the development of the local inflammatory changes and the systemic manifestations that characterize syphilis and Lyme disease.

A simple assay for proteolysis of IGFBP-3

Abstract: An assay for assessing the proteolysis of IGFBP-3 by a biological fluid or protein has been developed using 125I hIGFBP-3 recombinantly derived from Escherichia coli as a substrate indicator. The labelled substrate is incubated at 37° in the presence of the agent being tested and proteolysis is visualized by SDS-PAGE and autoradiography. The assay showed that the pregnancy associtatcd protease is Ca++ dependent. The substrate is also proteolyed by rat and mouse pregnancy sera, making the assay applicable to studies in those animal systems.

Structural dynamics and functional domains of the fur protein.

Abstract: Proteolytic enzymes were used to detect metal-induced conformational changes in the ferric uptake regulation (Fur) protein of Escherichia coli K12. Metal binding results in enhanced cleavage of the N-terminal region of Fur by trypsin and chymotrypsin. Activation of both trypsinolysis sensitivity and DNA binding have similar metal ion specificity and concentration dependencies, suggesting that the conformational change detected is required for operator DNA binding. Isolation and characterization of biochemically generated fragments of Fur as well as other data indicate that the N-terminal region is necessary for the interaction of the repressor with DNA and that a C-terminal domain is sufficient for binding to metal ions.

High density lipoprotein apolipoproteins mediate removal of sterol from intracellular pools but not from plasma membranes of cholesterol-loaded fibroblasts.

Abstract: Cultured cells possess high-affinity binding sites (receptors) for high density lipoprotein (HDL) that appear to mediate removal of excess intracellular cholesterol from cells. To examine the role of intact HDL apoproteins in receptor-mediated cholesterol removal, HDL3 apoproteins were digested with the proteolytic enzymes trypsin and pronase, and the residual particles were used in sterol efflux experiments. Protease treatment abolished the interaction of HDL3 with the 110-kd cell membrane protein postulated to represent the HDL receptor molecule, indicating that this interaction is mediated by HDL apoproteins rather than lipids. Compared with native HDL3 protease-modified HDL3 had a markedly reduced ability to selectively remove sterol from intracellular pools, even though modified particles promoted greater cholesterol efflux from the plasma membrane than did native particles. These results indicate that whereas sterol efflux from plasma membranes is mediated by HDL lipids, removal of excess intracellular sterol from cells is mediated by HDL apoproteins. These findings are consistent with the hypothesis that receptor binding of HDL apoproteins stimulates translocation of excess intracellular sterol to the cell surface where it becomes accessible for removal by HDL or other lipid-rich acceptor particles.

Preovulatory changes in ovarian expression of collagenases and tissue metalloproteinase inhibitor messenger ribonucleic acid: role of eicosanoids.

Abstract: The preovulatory surge of gonadotropins activates a cascade of proteolytic enzymes resulting in the rupture of the follicular wall and the release of a fertilizable ovum during ovulation. In the rat the process is initiated by a rise in follicular tissue-type plasminogen activator, produced predominantly in granulosa cells. Recent studies revealed a preovulatory increase in ovarian collagenolytic activity in vivo and an increase in activatable collagenase in vitro. In view of the complicated control of mammalian collagenase synthesis and activity by local inhibitors and activators, we examined the expression of ovarian interstitial and type IV collagenases and tissue inhibitor of metalloproteinase (TIMP) mRNA after an ovulatory stimulus. Ovarian mRNA was isolated from immature PMSG-treated rats 3, 6, and 9 h after hCG stimulation. Northern blot analyses revealed a mRNA of 1.7 kilobases (kb) hybridizing with the human interstitial collagenase cDNA probe. The levels of this mRNA showed a 25-fold increase between 3-6 h after hCG stimulation. The human cDNA probe of collagenase IV hybridized with a mRNA of 3.1 kb, which showed only a 4-fold increase 9 h after hCG treatment. The interstitial collagenase mRNA was expressed in both granulosa cells of preovulatory follicles and the residual ovarian tissue, whereas the expression of collagenase IV mRNA was limited to the residual tissue. Inhibitors of eicosanoid synthesis, previously shown to block ovulation and the LH/hCG-induced rise in ovarian collagenolysis, suppressed the gonadotropic stimulation of interstitial collagenase mRNA, but slightly stimulated that of collagenase IV. The mouse cDNA probe of TIMP hybridized with a 0.9-kb mRNA, which was stimulated by hCG to reach a maximum (7- to 8-fold increase) between 6-9 h after stimulation. TIMP was expressed and stimulated in both the granulosa cells and the residual tissue. Inhibitors of eicosanoid synthesis did not affect the gonadotropic stimulation of TIMP mRNA. These data support the suggested role of interstitial collagenase in follicle rupture and the essential role of eicosanoids in the mediation of gonadotropic stimulation of interstitial collagenase production and action. The observed stimulation of TIMP mRNA expression by the gonadotropin and the lack of any effect of eicosanoid synthesis inhibitors on this action of LH/hCG offer an additional mechanism by which these inhibitors may block ovulation. Thus, the suppression of ovulation by inhibitors of eicosanoid synthesis may result from selective inhibition of interstitial collagenase expression and undisturbed gonadotropin-stimulated TIMP expression.

Novel enzymatically degradable polymers comprising α-amino acid, 1,2-ethanediol, and adipic acid

Abstract: Novel water-insoluble polymers, which are degraded and solubilized by the action of specific proteolytic enzymes, were successfully synthesized from α-amino acid, 1,2-ethanediol, and adipic acid.

Casein utilization by lactococci.

Abstract: Mixed starter cultures used to manufacture Dutch cheeses include Lactococcus and Leuconostoc species, the former being quantitatively more important (3, 13). Several metabolic properties of lactococci serve special functions which directly or indirectly have an impact on processes such as flavor development and ripening of the cheese (31). These functions are (i) fermentation and depletion of the milk sugar lactose, (ii) reduction of the redox potential of the cheese, (iii) citrate fermentation, and (iv) casein degradation. Casein degradation by lactococci yields free amino acids, as well as lowand high-molecular-weight peptides. These are the sources of essential and growth-stimulating amino acids for the members of the genus Lactococcus (42). Besides being an important nutritional source for the starter bacteria, casein also plays a crucial role in the development of flavor in cheese. Certain peptides contribute to the formation of a typical cheese flavor, and other, undesirable bitter-tasting peptides can be produced (39, 45, 47). A better understanding of the processes leading to the formation of these flavor peptides requires knowledge about all enzymes involved in casein degradation. The sequential steps involved in ,-casein degradation and the cellular location of the enzymes involved will be reviewed in this paper. Special attention will be paid to the role of amino acid and peptide transport systems in the overall process of casein degradation and the overall growth performance of the organism. On the basis of the proteolytic breakdown products of casein and the substrate specificities of the peptidases and transport systems, a minimum model for the growth of lactococci in casein-containing media is proposed.

High-purity isolation of bullfrog hair bundles and subcellular and topological localization of constituent proteins.

Abstract: The small number of hair cells in auditory and vestibular organs severely impedes the biochemical characterization of the proteins involved in mechano-electrical transduction. By developing an efficient and clean "twist-off" method of hair bundle isolation, and by devising a sensitive, nonradioactive method to detect minute quantities of protein, we have partially overcome this limitation and have extensively classified the proteins of the bundles. To isolate hair bundles, we glue the saccular macula of the bullfrog to a glass coverslip, expose the tissue to a molten agarose solution, and allow the agarose to solidify to a firm gel. By rotating the gel disk with respect to the fixed macula, we isolate the hair bundles by shearing them at their mechanically weak bases. The plasma membranes of at least 80% of the stereocilia reseal. To visualize the proteins of the hair bundle, we covalently label them with biotin, separate them by SDS-PAGE, and transfer them to a charged nylon membrane. We can detect less than 500 fg of protein by probing the membrane with streptavidin-alkaline phosphatase and detecting the chemiluminescent product from the hydrolysis of the substrate 3-(4-methoxyspiro-(1,2-dioxetane-3,2'-tricyclo-[3.3.1. 1(3.7)]decan)-4-yl) phenyl phosphate (AMPPD). These techniques reveal a distinct constellation of proteins in and associated with hair bundles. Several proteins, such as calmodulin, calbindin, actin, tubulin, and fimbrin, have previously been described. A second class of proteins in the preparation appears to be derived from extracellular sources. Finally, several heretofore undescribed bundle proteins are identified and characterized by their membrane topology, subcellular localization, and glycosidase and protease sensitivities.

Effect of long-term cigarette smoke exposure on pulmonary vascular structure and function in the guinea pig.

Abstract: To investigate the effect of chronic exposure to cigarette smoke on the structure and function of the pulmonary vasculature, we used a guinea pig model of cigarette smoke-induced emphysema, in which groups of guinea pigs were exposed to smoke for periods of 1, 3, 6, and 12 months. We found that the mean pulmonary artery pressure in smokers was increased at one month, a time at which there was no evidence of emphysema. Although the vascular pressures remained elevated, they did not progressively increase, even though there was progressive lung destruction. Pulmonary hypertension was associated with muscularization of the arterioles, seen as an increase in the percentage of small pulmonary vessels with double elastic lamina. We conclude that chronic exposure to cigarette smoke will produce pulmonary hypertension in the guinea pig, and that the hemodynamic changes are accompanied by alteration of the structure of the small pulmonary arterioles and arteries. The apparent dissociation of pulmonary hypertension and emphysema suggests that the pulmonary hypertension is not due to destruction of the lung capillary bed. The etiology of this process may be smoke-induced inflammation with release of vasoactive substances as well as proteolytic enzymes.

Principles of stopped-flow sequential injection analysis and its application to the kinetic determination of traces of a proteolytic enzyme.

Abstract: The concept of sequential injection analysis is described, and an application of this novel technique to an assay of traces of a proteolytic enzyme is demonstrated. The automated method is based on a stopped-flow procedure with fluorescence detection. Instead of using the conventional constant flow rate, a sinusoldal flow pattern, as generated by a robust cam-driven piston pump, has been applied. The validation of the novel flow injection system using a nonreactive fluorescent compound yielded highly reproducible results over a concentration range of 3 order of magnitude. The calibration graph for the enzyme activities in the range 5-75 micron mL-1 could be fitted a second-order polynomial. The detection limit is 2.5 micron mL-1, which corresponds to 7.2 ng mL-1 pure active enzyme.