Detection Of Proteolytic Activity Research Articles

MMP-9 release into collateral blood vessels before endovascular thrombectomy to assess the risk of major intracerebral haemorrhages and poor outcome for acute ischaemic stroke: a proof-of-concept study

Matrix metalloproteinases (MMPs) are implied in blood-brain barrier degradation and haemorrhagic transformation following ischaemic stroke, but their local relevance in the hyperacute disease phase is unknown. We aimed to examine ultra-early MMP-9 and MMP-2 release into collateral blood vessels, and to assess its prognostic value before therapeutic recanalisation by endovascular thrombectomy (EVT). We report a cross-sectional proof-of-concept study including patients undergoing EVT for large-vessel ischaemic stroke at the University Hospital Würzburg, Germany. We obtained liquid biopsies from the collateral circulation before recanalisation, and systemic control samples. Laboratory workup included quantification of MMP-9 and MMP-2 plasma concentrations by cytometric bead array, immunohistochemical analyses of cellular MMP-9 and MMP-2 expression, and detection of proteolytic activity by gelatine zymography. The clinical impact of MMP concentrations was assessed by stratification according to intracranial haemorrhagic lesions on postinterventional computed tomography (Heidelberg Bleeding Classification, HBC) and early functional outcome (modified Rankin Scale, mRS). We used multivariable logistic regression, receiver-operating-characteristic (ROC) curves, and fixed-level estimates of test accuracy measures to study the prognostic value of MMP-9 concentrations. Between August 3, 2018, and September 16, 2021, 264 matched samples from 132 patients (86 [65.2%] women, 46 [34.8%] men, aged 40-94 years) were obtained. Median (interquartile range, IQR) MMP-9 (279.7 [IQR 126.4-569.6] vs 441 [IQR 223.4-731.5] ng/ml, p<0.0001) but not MMP-2 concentrations were increased within collateral blood vessels. The median MMP-9 expression level of invading neutrophils was elevated (fluorescence intensity, arbitrary unit: 2276 [IQR 1007-5086] vs 3078 [IQR 1108-7963], p=0.0018). Gelatine zymography experiments indicated the locally confined proteolytic activity of MMP-9 but not of MMP-2. Pretherapeutic MMP-9 release into stroke-affected brain regions predicted the degree of intracerebral haemorrhages and clinical stroke severity after recanalisation, and independently increased the odds of space-occupying parenchymal haematomas (HBC1c-3a) by 1.54 times, and the odds of severe disability or death (mRS ≥5at hospital discharge) by 2.33 times per 1000ng/ml increase. Excessive concentrations of MMP-9 indicated impending parenchymal haematomas and severe disability or death with high specificity. Measurement of MMP-9 within collateral blood vessels is feasible and identifies patients with stroke at risk of major intracerebral haemorrhages and poor outcome before therapeutic recanalisation by EVT, thereby providing evidence of the concept validity of ultra-early local stroke biomarkers. This work was funded by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) and the Interdisciplinary Centre for Clinical Research (IZKF) at the University of Würzburg.

Tetrameric Antibody Complexes and Affinity Tag Peptides for the Selective Immobilization and Imaging of Single Quantum Dots.

Colloidal semiconductor quantum dots (QDs) are of widespread interest as fluorescent labels for bioanalysis and imaging applications. Single-particle measurements have proven to be a very powerful tool for better understanding the fundamental properties and behaviors of QDs and their bioconjugates; however, a recurring challenge is the immobilization of QDs in a solution-like environment that minimizes interactions with a bulk surface. Immobilization strategies for QD-peptide conjugates are particularly underdeveloped within this context. Here, we present a novel strategy for the selective immobilization of single QD-peptide conjugates using a combination of tetrameric antibody complexes (TACs) and affinity tag peptides. A glass substrate is modified with an adsorbed layer of concanavalin A (ConA) that binds a subsequent layer of dextran that minimizes nonspecific binding. A TAC with anti-dextran and anti-affinity tag antibodies binds to the dextran-coated glass surface and to the affinity tag sequence of QD-peptide conjugates. The result is spontaneous and sequence-selective immobilization of single QDs without any chemical activation or cross-linking. Controlled immobilization of multiple colors of QDs is possible using multiple affinity tag sequences. Experiments confirmed that this approach positions the QD away from the bulk surface. The method supports real-time imaging of binding and dissociation, measurements of Förster resonance energy transfer (FRET), tracking of dye photobleaching, and detection of proteolytic activity. We anticipate that this immobilization strategy will be useful for studies of QD-associated photophysics, biomolecular interactions and processes, and digital assays.

Detection of proteolytic activity by covalent tethering of fluorogenic substrates in zymogram gels.

Current zymographic techniques detect only a subset of known proteases due to the limited number of native proteins that have been optimized for incorporation into polyacrylamide gels. To address this limitation, we have developed a technique to covalently incorporate fluorescently labeled, protease-sensitive peptides using an azido-PEG3-maleimide crosslinker. Peptides incorporated into gels enabled measurement of MMP-2, -9, -14, and bacterial collagenase. Sensitivity analysis demonstrated that use of peptide functionalized gels could surpass detection limits of current techniques. Finally, electrophoresis of conditioned media from cultured cells resulted in the appearance of several proteolytic bands, some of which were undetectable by gelatin zymography. Taken together, these results demonstrate that covalent incorporation of fluorescent substrates can greatly expand the library of detectable proteases using zymographic techniques.

In Situ Demonstration and Characteristic Analysis of the Protease Using Substrate Immersing Zymography.

Zymography, the detection of proteolytic activities on the basis of protein substrate degradation, has been a technique described in the literature for at least in the past 50 years. In this study, we used substrate immersing zymography to analyze proteolysis of proteases. Instead of being directly added into a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, the substrates were added into the immersing solution after electrophoresis. With substrate immersing zymography, some characters of proteases, such as enzyme forms, potential proteolytic activity, molecular weights, presence of complexes, and potentially active enzyme fragments in complex biological samples, can be determined.

Water-soluble proteinase activity of gastric fluids in the European whip snake, Hierophis viridiflavus : an experimental preliminary survey

Understanding the mechanism of metabolic digestion and of food assimilation in wild snake species is of fundamental importance to compare trophic niches, their adaptive significance and evolutionary patterns of diet selection for different snake species. From two adult Hierophis viridiflavus males in fasting condition, we sampled gastric fluids using fiberoptic endoscopy. We analyzed the proteolytic activities (optimal pH and temperature, response to inhibitors) of snake samples by zymography techniques. The two major protease activity bands were always detected at every tested pH. Activity bands were also detectable over 37 °C; in particular both bands showed the highest activity ranging from 55 °C to 60 °C. Ranging from 65 °C to 85 °C, one band remained visible while the other band disappeared over 60 °C. The pattern of proteolytic enzymes activity of sampled gastric fluids highlighted a scenario composed of few active proteases, reflecting the feeding status (e.g., fasting) of studied snakes. Detection of proteolytic activity until 85 °C, as in prokaryotic organisms, supported the hypothesis about the presence of proteases of exogenous source.

Virus-Induced Chaperone-Enriched (VICE) Domains Function as Nuclear Protein Quality Control Centers during HSV-1 Infection

Virus-Induced Chaperone-Enriched (VICE) domains form adjacent to nuclear viral replication compartments (RC) during the early stages of HSV-1 infection. Between 2 and 3 hours post infection at a MOI of 10, host protein quality control machinery such as molecular chaperones (e.g. Hsc70), the 20S proteasome and ubiquitin are reorganized from a diffuse nuclear distribution pattern to sequestration in VICE domains. The observation that VICE domains contain putative misfolded proteins suggests that they may be similar to nuclear inclusion bodies that form under conditions in which the protein quality control machinery is overwhelmed by the presence of misfolded proteins. The detection of Hsc70 in VICE domains, but not in nuclear inclusion bodies, indicates that Hsc70 is specifically reorganized by HSV-1 infection. We hypothesize that HSV-1 infection induces the formation of nuclear protein quality control centers to remodel or degrade aberrant nuclear proteins that would otherwise interfere with productive infection. Detection of proteolytic activity in VICE domains suggests that substrates may be degraded by the 20S proteasome in VICE domains. FRAP analysis reveals that GFP-Hsc70 is dynamically associated with VICE domains, suggesting a role for Hsc70 in scanning the infected nucleus for misfolded proteins. During 42°C heat shock, Hsc70 is redistributed from VICE domains into RC perhaps to remodel viral replication and regulatory proteins that have become insoluble in these compartments. The experiments presented in this paper suggest that VICE domains are nuclear protein quality control centers that are modified by HSV-1 to promote productive infection.

Metallopeptidases produced by group B Streptococcus: Influence of proteolytic inhibitors on growth and on interaction with human cell lineages

Group B Streptococcus (GBS) is a major etiologic agent of neonatal bacterial infections and is the most common cause of sepsis and pneumonia in newborns. Surface and secreted molecules of GBS are often essential virulence factors which are involved in the adherence of the bacteria to host cells or are required to suppress the defense mechanisms of hosts. We analyzed the peptidase profiles of GBS by detection of proteolytic activities on SDS-PAGE containing copolymerized gelatin as substrate. Based on the inhibition by o-phenathroline and EGTA, three distinct peptidases of 220, 200 and 180 kDa were identified in the culture medium, besides one major cell-associated proteolytic activity, a 200-kDa metallopeptidase, suggesting that all were zinc-metallopeptidases. GBS culture supernatants, rich in metallotype peptidases, also cleaved fibronectin, laminin, type IV collagen, fibrinogen and albumin. Cleavage of the host extracellular matrix by GBS may be a relevant factor in the process of bacterial dissemination and/or invasion. Notably, metallopeptidase inhibitors strongly blocked GBS growth as well as its interaction with human cell lineages. Understanding the contribution of peptidases to the pathogenesis of GBS disease may broaden our perception of how this significant pathogen causes severe infections in newborn infants.

Detección de proteasas extracelulares en especies de Mucor

The fungi are characterized by their abilities to produce and secrete enzymes to the external environment. The species of genus Mucor are a group of fungal microbes with important biotechnological potential, which are responsible for production of industrial enzymes. This work evaluated the ability of protease production in twelve species of genus Mucor. The strains were kept for 120 h under the incubation temperature of 28 degrees C on a shaker at 120 rpm. The detection of proteolytic activity was evaluated in all species, the higher activity was detected in Mucor racemosus Fres. f. chibinensis (Neophytova) Schipper.

Detection of proteolytic activity by fluorescent zymogram in-gel assays.

Proteases are involved in the regulation of many biological functions. This study describes a novel method for detecting protease activity by fluorescent zymogram in-gel protease assays, using SDS polyacrylamide gels copolymerized with a peptide-MCA (4-methyl-coumaryl-7-amide) substrate. This method allows simultaneous determination of protease cleavage specificity and molecular weight. Trypsin was electrophoresed in SDS polyacrylamide gels copolymerized with Boc-Gln-Ala-Arg-MCA, the gel was then incubated in assay buffer, and trypsin cleavage of the peptide-MCA substrate generated fluorescent AMC (7-amino-4-methyl-coumarin), which was subsequently detected under UV transillumination. Chymotrypsin activity was detected in gels copolymerized with Suc-Ala-Ala-Pro-Phe-MCA substrate. Selective detection of these proteases was demonstrated by the absence of trypsin activity in gels containing the chymotrypsin substrate, and the lack of chymotrypsin activity in gels containing the trypsin substrate. Detection of proteolytic activity from secretory vesicles of adrenal medulla (chromaffin granules) was observed with the trypsin substrate, Z-Phe-Arg-MCA, but not with the chymotrypsin substrate. Overall, this sensitive fluorescent zymogram in-gel protease assay method can be used for rapid determination of protease cleavage specificity and enzyme molecular weight in biological samples. This assay should be useful for many research disciplines investigating the role of the many proteases that control cellular functions.

Detection of cysteine-proteinases in Leishmania amazonensis promastigotes using a cross-reactive antiserum

Rabbit serum against the cysteine-proteinases papain has been employed for the cellular localization of cysteine-proteinases of in Leishmania amazonensis promastigotes. By immunocytochemistry, immune complexes were found in the plasma membrane and in the flagella pocket of the parasite. The antiserum immunoprecipitated major iodinated proteins with molecular masses of 66, 45, 28 and 24 kDa and a wide partitioning of the Triton X-114 detergent phase. The presence of cysteine-proteinase at the cell surface membrane was also suggested by the detection of proteolytic activity in living cells (19.0 μg azocasein min −1 10 −7 promastigotes (1.0 S.D.)).

Detection of cysteine-proteinases in Leishmania amazonensis promastigotes using a cross-reactive antiserum.

Rabbit serum against the cysteine-proteinases papain has been employed for the cellular localization of cysteine-proteinases of in Leishmania amazonensis promastigotes. By immunocytochemistry, immune complexes were found in the plasma membrane and in the flagella pocket of the parasite. The antiserum immunoprecipitated major iodinated proteins with molecular masses of 66, 45, 28 and 24 kDa and a wide partitioning of the Triton X-114 detergent phase. The presence of cysteine-proteinase at the cell surface membrane was also suggested by the detection of proteolytic activity in living cells (19.0 microg azocasein min(-1) 10(-7) promastigotes (1.0 S.D. )).

Prochymosin polymorphism in calves of black-and-white cattle and their crosses with Simental bulls.

Polymorphism of prochymosin was observed in individual calf abomasa, using agarose gel electrophoresis followed by detection of proteolytic activity. Abomasum samples were randomly collected during slaughtering from 239 and 146 calves (3-5 weeks old) of Black-and-White cattle and their crosses with Simental bulls, respectively. Four distinct prochymosins were found and, according to their decreasing electrophoretic mobility in alkaline agarose gel, termed as prochymosin A, D, B and C which occurred singly and in pairs (then with equal proteolytic activities of both components). Prochymosin A, B and C (designation according to FOLTMANN, 1966) activated at pH 4.7 was transformed into electrophoretically distinct chymosin. When prochymosin D was activated at this pH, chymosin D showed similar mobility as chymosin B both at alkaline and acidic pHs. Prochymosin variants occurred at genetical equilibrium in nine and ten phenotypes in the first and second genetic group. The distribution of phenotypes in the two groups differed significantly (P < 0.05). The gene frequencies of prochymosin A, D, B and C were 0.35, 0.11, 0.52 and 0.02 in Black-and-White calves, and 0.39, 0.08, 0.47 and 0.06 in crosses, respectively. These prochymosins were controlled by four pairs of codominant alleles. A possible correlation of the results obtained by FOLTMANN (1966) with ours and those of ASATO and RAND (1972, 1977) was discussed.

A new method for the detection of proteolytic activity in Pseudomonas lundensis after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis

A new method for the visualization of proteolytic activity in cell culture supernatant from Pseudomonas lundensis after sodium dodecyl sulfate (SDS)--gel electrophoresis is described. Following conventional electrophoresis, the gel is washed in a methanol-containing buffer to facilitate partial removal of SDS. After incubation with 0.5% casein the gel is stained for protein with Coomassie Brilliant Blue R-250. Bands with proteolytic activity appear as clear areas in the gel against a blue-stained background. Molecular weight standards electrophoresed in the same gel stain more intensely than the background and allow determination of the molecular weights of the proteolytic components. The sensitivity of post-electrophoretic reactivation in SDS-gels was determined using trypsin as standard. A slight modification of the technique allowed detection of proteolytic activity in nondenaturing and in isoelectric focusing gels.

Occurrence of prochymosin variants in the abomasum of bovine foetuses and calves.

Prochymosin variants were analysed in single abomasa from 67 foetuses (3-9 months of gestation) and 33 calves (about 6 weeks old) of Black-and-White cattle collected in a slaughter house. The method of agarose gel electrophoresis followed by detection of proteolytic activity was used. Three distinct prochymosins A, B and C that occurred singly or in pairs (with equal proteolytic activities of both components) were found. Chymosin A, B and C obtained after conversion of corresponding prochymosins, demonstrated similar electrophoretical mobilities like the three chymosin fractions contained in commercial rennin (Sigma, USA). Our chymosin B showed identical mobility as the amidated form of recombined chymosin B contained in Chymogen (Chr. Hansen's Lab., Denmark A/S). Prochymosin A, B and C in the examined animals were precursors of corresponding chymosins and were controlled by three separate codominant alleles. The following prochymosin phenotypes were found: AA (30), AB (32), AC (4), BB (29), BC (4) and CC (1). Chi-square analysis demonstrated significant differences between the observed and expected numbers of phenotypes. The gene frequencies of prochymosin A, B and C were 0.48, 0.47 and 0.05, respectively.

45] Zymographic techniques for detection and characterization of microbial proteases

We have presented a variety of zymographic techniques for identification and characterization of microbial proteases, using SDS-PAGE and PAGE in nondissociating gels. Techniques are described using copolymerized protein substrates, diffusable protein substrates, protein substrates incorporated into indicator gels, as well as synthetic esterase substrates. When a newly discovered protease is being characterized, it is advisable to try a variety of techniques, both to determine optimal conditions for enzyme detection and to characterize the protease. Zymography is a versatile two-stage technique involving protein separation by electrophoresis followed by detection of proteolytic activity. Each particular combination of protease separation and detection techniques had advantages and limitations. Protease separation by SDS-PAGE has as a limitation the fact that some proteases do not renature and hence cannot be detected following treatment with SDS. However, it has an advantage the fact that it allows estimation of the relative molecular weight of proteases. Protein separation using nondissociating PAGE is performed using much gentler protease inactivation conditions than those produced by treatment with SDS. Like SDS-PAGE, nondissociating PAGE permits detection of multiple forms of enzymes; however, a disadvantage is that it cannot be used to obtain molecular weight estimates of proteases. The main variable to control during development of zymograms is the length of time of incubations. Increasing incubation (development) time generally increases the sensitivity of protease detection; however, as the length of time of incubation increases so does the extent of diffusion of proteases and substrates. If incubations are prolonged, protease bands will diffuse, decreasing resolution. Additionally, zones of lysis produced by closely migrating proteolytically active species will merge, eliminating the possibility of detecting all proteolytic species in the sample. Zymographic techniques can be extremely useful in identification and characterization of microbial proteases. If a few properties of a protease are known, such as the pH range over which the enzyme is active, and whether it can renature after exposure to SDS, zymographic techniques can be specifically and readily adapted to optimize conditions for detection and assist in characterization of the enzyme.

Crossed immunoelectrophoretic analysis of proteins from Antarctic krill (Euphausia superba) with special reference to serine proteinases

Immunochemical characterization of proteins from Antarctic krill (E. superba) and particularly serine proteinases was performed by crossed immunoelectrophoresis (CIE). A reference pattern with 34 arbitrarly numbered immunoprecipitates was established for a crude krill extract using IgG rabbit antiserum. A zymographic technique for detection of proteolytic activity was applied and revealed 4 casein-positive antigens. Introduction of different affinity media as intermediate gels in CIE demonstrated several proteins which strongly interacted with Sepharose-bound arginine and Con A. These results suggest the presence of trypsin-like enzymes and other serine proteases and a high content of glycoproteins with alfa-D-mannopyranoside and alfa-D-glycopyranoside residues. The CIE method thus offers a reliable and reproducible tool for identification and characterization of different krill enzymes as well as application in process control, when purifying individual proteins/enzymes. Moreover this technology may serve as a fingerprinting method suitable for biological studies such as classification of geographical subpopulations, genotyping a specific species as well as comparisons between different species.

Growth of an extracellular proteinase-deficient strain of Pseudomonas fluorescens on milk and milk proteins

An extracellular proteinase-and lipase-deficient mutant of a psychrotroph, Pseudomonas fluorescens strain 32A, has been isolated and the absence of the proteinase enzyme confirmed by growth on differential media, enzyme assay and polyacrylamide gel electrophoresis. Competition between the parent and the mutant was observed when equal numbers of the 2 strains were inoculated together into raw skim-milk at 6 degrees C. Bitterness was detected at 6 degrees C in pasteurized skim-milk inoculated with the parent cells concurrent with the detection of proteolytic activity. In the case of the mutant, slight bitterness which did not increase with increasing cell numbers was detected in the absence of proteolysis. Mutant cells failed to grow on Na caseinate as the sole source of carbon. It was concluded that the extracellular proteinase, while not essential for growth in milk, does provide a selective advantage to the producer organism. This enzyme is, however, essential for growth on milk proteins and contributes to the development of bitterness in pasteurized milk.

Detection of proteolytic activity after isoelectric focusing in polyacrylamide gel

δ-Chymotrypsin, subtilopeptidase and crude extracellular proteases from Pseudomonas aeruginosa have been separated by isoelectric focusing in thin layers of polyacrylamide gel. Ampholine was removed by soaking the gel in buffer, prior to overlaying with casein-containing agar to detect proteolysis. The technique described is simple and has a high resolving power. It should have general application for the analysis of enzymes.

Immunoelectrophoretic characterization of proteolytic enzymes.

THE detection of proteolytic activity after electro-phoresis in agar gel has been described previously1,2. It has been shown with a number of hydrolytic enzymes3 that the precipitation lines formed by the antigen–antibody reaction after immunoelectrophoresis in agar gel exhibit enzymatic activity. This enzymatic activity can be demonstrated qualitatively after washing and drying the slides in the usual way by utilizing conventional histochemical techniques.