Activatable endoplasmic reticulum-targeted NIR fluorescent probe with a large Stokes shift for detecting and imaging chymotrypsin.

In this work, the first endoplasmic reticulum-targeted near-infrared fluorescent probe, ISO-Chy, with a dicyanoisophorone derivative as a fluorophore is reported by introducing the recognition group of 4-bromobutyl for chymotrypsin detection. The probe can be easily synthesized and has shown satisfactory sensitivity and selectivity to chymotrypsin. Meanwhile, ISO-Chy has a large Stokes shift (135 nm) to minimize self-absorption and interference from autofluorescence and then generate significant fluorescence enhancement upon incubation with chymotrypsin. Additionally, ISO-Chy has an excellent ability to target the endoplasmic reticulum, along with preferable Pearson's correlation coefficients (Rr) of 0.9411 and 0.9522 in P815 cells and HepG2 cells, respectively. Moreover, ISO-Chy was successfully utilized to visualize endogenous chymotrypsin in P815 cells and HepG2 cells and was first used to detect chymotrypsin activity in HepG2 tumor-bearing mice. These findings indicate that ISO-Chy could be an effective tool for detecting endogenous chymotrypsin activity, supporting its use for investigating chymotrypsin function in pathologic processes.

Detection of Chymotrypsin by Optical and Acoustic Methods.

Chymotrypsin is an important proteolytic enzyme in the human digestive system that cleaves milk proteins through the hydrolysis reaction, making it an interesting subject to study the activity of milk proteases. In this work, we compared detection of chymotrypsin by spectrophotometric dynamic light scattering (DLS) and quartz crystal microbalance (QCM) methods and determined the limit of chymotrypsin detection (LOD), 0.15 ± 0.01 nM for spectrophotometric, 0.67 ± 0.05 nM for DLS and 1.40 ± 0.30 nM for QCM methods, respectively. The sensors are relatively cheap and are able to detect chymotrypsin in 3035 min. While the optical detection methods are simple to implement, the QCM method is more robust for sample preparation, and allows detection of chymotrypsin in non-transparent samples. We give an overview on methods and instruments for detection of chymotrypsin and other milk proteases.

A Molecular Probe with Both Chromogenic and Fluorescent Units for Detecting Serine Proteases.

A molecular probe with l-phenylalanine p-nitroanilide and l-lysin 4-methylcoumaryl-7-amide, in which these amino acid derivatives are connected through a succinic-acid spacer, was prepared. Trypsin and papain were detected by blue-fluorescence emission of generated 7-amino-4-methylcoumarin (AMC). α-Chymotrypsin and nattokinase were detected from both the blue-fluorescence emission of AMC and the UV absorbance of p-nitroaniline. In addition, different time courses of p-nitroaniline and AMC were observed between the reaction of P1 with α-chymotrypsin and that with nattokinase. In the case of nattokinase, both the fluorescence emission and UV absorbance slowly increased. In contrast, the increasing UV absorbance was saturated at the early stage of the reaction of the present probe with chymotrypsin, whereas the fluorescence emission continuously increased in the following stages.

Intracellular compartment-specific proteasome dysfunction in postmortem cortex in schizophrenia subjects.

Protein homeostasis is an emerging component of schizophrenia (SZ) pathophysiology. Proteomic alterations in SZ are well-documented and changes in transcript expression are frequently not associated with changes in protein expression in SZ brain. The underlying mechanism driving these changes remains unknown, though altered expression of ubiquitin proteasome system (UPS) components have implicated protein degradation. Previous studies have been limited to protein and transcript expression, however, and do not directly test the function of the proteasome. To address this gap in knowledge, we measured enzymatic activity associated with the proteasome (chymotrypsin-, trypsin-, and caspase-like) in the superior temporal gyrus (STG) of 25 SZ and 25 comparison subjects using flourogenic substrates. As localization regulates which cellular processes the proteasome contributes to, we measured proteasome activity and subunit expression in fractions enriched for nucleus, cytosolic, and membrane compartments. SZ subjects had decreased trypsin-like activity in total homogenate. This finding was specific to the nucleus-enriched fraction and was not associated with changes in proteasome subunit expression. Interestingly, both chymotrypsin-like activity and protein expression of 19S RP subunits, which facilitate ubiquitin-dependent degradation, were decreased in the cytosol-enriched fraction of SZ subjects. Intracellular compartment-specific proteasome dysfunction implicates dysregulation of protein expression both through altered ubiquitin-dependent degradation of cytosolic proteins and regulation of protein synthesis due to degradation of transcription factors and transcription machinery in the nucleus. Together, these findings implicate proteasome dysfunction in SZ, which likely has a broad impact on the proteomic landscape and cellular function in the pathophysiology of this illness.

Nutritional markers in patients with diabetes and pancreatic exocrine failure.

AIMS: Altered pancreatic exocrine function can be observed in patients with type 1 or type 2 diabetes. In the present study, we evaluated the potential nutritional consequences of this dysfunction. METHODS: Serum concentrations of nutritional markers, including albumin, cholesterol, triacylglycerol, vitamins A, D, and E, were assessed in a cohort of 468 patients (137 with type 1 diabetes and 331 with type 2 diabetes), after exclusion of the patients with a CRP > 10 mg/l. These patients were compared with 47 patients with diseases of the exocrine pancreas and diabetes (type 3c diabetes or pancreatogenic diabetes). Fecal elastase-1 and chymotrypsin concentrations were measured and patients with type 1 and type 2 diabetes were divided into three groups according to whether zero (group NN), one (group LN), or both (group LL) concentrations were decreased. RESULTS: Several markers differed significantly between the groups of patients, including BMI, albumin, phosphorus, and fat-soluble vitamins. Patients with pancreatogenic diabetes had markedly more profound alterations than patients with type 1 or type 2 diabetes and altered exocrine function. However, patients with type 1 or type 2 diabetes and decreased concentrations of both elastase-1 and chymotrypsin had lower albumin, phosphorus, and vitamin A than patients with normal pancreatic exocrine function. CONCLUSIONS: Modest nutritional alterations were found in patients with type 1 or type 2 diabetes and altered exocrine function. Patients with type 1 or type 2 diabetes and altered exocrine function may thus deserve to be screened for nutritional deficiencies.

Proteinase pattern of honeybee prepupae from healthy and American Foulbrood infected bees investigated by zymography.

American foulbrood disease (AFB) is the main devastating disease that affects honeybees' brood, caused by Paenibacillus larvae. The trend of the research on AFB has addressed the mechanisms by which P. larvae bacteria kill honeybee larvae. Since prepupae could react to the infection of AFB by increasing protease synthesis, the aim of this work was to compare protease activity in worker prepupae belonging to healthy colonies and to colonies affected by AFB. This investigation was performed by zymography. In gel, proteolytic activity was observed in prepupae extracts belonging only to the healthy colonies. In the prepupae extracts, 2D zimography followed by protein identification by MS allowed to detect Trypsin-1 and Chymotrypsin-1, which were not observed in diseased specimens. Further investigations are needed to clarify the involvement of these proteinases in the immune response of honeybee larvae and the mechanisms by which P. larvae inhibits protease production in its host.

A novel three-fluorophore system as a ratiometric sensor for multiple protease detection.

A synthetic three-fluorophore system with two enzymatically cleavable linkers has been developed for the simultaneous detection of two proteases in a mixture. The probe was designed to afford single excitation/triple emission ratiometric detection through a fluorescence change during the cleavage of a peptide linker. The developed assays were verified for trypsin and chymotrypsin as the model enzymes.

Transfer Zymography.

The technique described here, transfer zymography, was developed to overcome two limitations of conventional zymography. When proteolytic enzymes are resolved by nonreducing SDS-PAGE into a polyacrylamide gel with copolymerized protein substrate, the presence of the protein substrate can result in anomalous, often slower, migration of the protease and an estimated mass higher than its actual mass. A further drawback is that the presence of a high background of substrate protein interferes with proteomic analysis of the protease band by excision, tryptic digestion, and LC-MS/MS analysis. In transfer zymography, the proteolytic enzymes are resolved by conventional nonreducing SDS-PAGE, without protein substrate in the gel. The proteins in the resolving gel are then electrophoretically transferred to a receiving gel that contains the protein substrate, by a process similar to western blotting. The receiving gel is then processed in a manner similar to conventional zymography. SDS is removed by Triton X-100 and incubated in conditions suitable for the proteolytic activity. After protein staining, followed by destaining, bands representing regions with active protease are visualized as clear bands in a darkly stained background. For proteomic analysis, electrophoresis is carried out simultaneously on a second resolving gel, and the bands corresponding to the clear regions in the receiving gel after zymogram development are excised for proteomic analysis.

Nonpeptide-Based Small-Molecule Probe for Fluorogenic and Chromogenic Detection of Chymotrypsin.

We report herein a nonpeptide-based small-molecule probe for fluorogenic and chromogenic detection of chymotrypsin, as well as the primary application for this probe. This probe was rationally designed by mimicking the peptide substrate and optimized by adjusting the recognition group. The refined probe 2 exhibits good specificity toward chymotrypsin, producing about 25-fold higher enhancement in both the fluorescence intensity and absorbance upon the catalysis by chymotrypsin. Compared with the most widely used peptide substrate (AMC-FPAA-Suc) of chymotrypsin, probe 2 shows about 5-fold higher binding affinity and comparable catalytical efficiency against chymotrypsin. Furthermore, it was successfully applied for the inhibitor characterization. To the best of our knowledge, probe 2 is the first nonpeptide-based small-molecule probe for chymotrypsin, with the advantages of simple structure and high sensitivity compared to the widely used peptide-based substrates. This small-molecule probe is expected to be a useful molecular tool for drug discovery and chymotrypsin-related disease diagnosis.

Fish condensate as effective replacer of fish meal protein in diet for striped snakehead, Channa striata (Bloch).

The optimal protein replacement of fish meal (FM) by fish condensate (FC) was investigated in striped snakehead, Channa striata (Bloch) (1.78 ± 0.02 g initial weight). The FM-based diet (0FC) was replaced by substituting protein from FC for 100 (100FC), 200 (200FC), 300 (300FC), 400 (400FC), 500 (500FC) or 600 (600FC) g kg-1 of the FM, and a commercial diet (CD) for carnivorous fish was included for comparison. The experiment was conducted indoors under completely randomized design (8 treatments × 3 replications × 60 fish per pond) over a 6-month trial. There were no significant differences in water quality during the experiment. The fish fed with 500FC had superior growth performance and feed utilization. This dietary treatment gave similar levels to all observed specific activities of digestive enzymes as did baseline 0FC. Survival, carcass composition, hematological parameters and liver histopathology were not negatively impacted by this protein replacement level. Economic analysis also supports the use of this by-product as a potent protein replacer in striped snakehead diet. Findings from the current study indicate that a 500 g kg-1 protein replacement of FM by FC is near optimal for striped snakehead, and similar use of it in the aquafeed of other species appears worth further studies.

Direct Observation of Kinetic Pathways of Biomolecular Recognition.

The pathways of molecular recognition, which is a central event in all biological processes, belong to the most important subjects of contemporary research in biomolecular science. By using fluorescence spectroscopy in a microfluidics channel, it can be determined that molecular recognition of α-chymotrypsin in hydrous surroundings at two different pH values (3.6 and 6.3) follows two distinctly different pathways. Whereas one corroborates an induced-fit model (pH 3.6), the other one (pH 6.3) is consistent with the selected-fit model of biomolecular recognition. The role of massive structural perturbations of differential recognition pathways could be ruled out by earlier XRD studies, rather was consistent with the femtosecond-resolved observation of dynamic flexibility of the protein at different pH values. At low concentrations of ligands, the selected-fit model dominates, whereas increasing the ligand concentration leads to the induced-fit model. From molecular modelling and experimental results, the timescale associated with the conformational flexibility of the protein plays a key role in the selection of a pathway in biomolecular recognition.

Bowman-Birk proteinase inhibitor from Clitoria fairchildiana seeds: Isolation, biochemical properties and insecticidal potential.

Herein described is the biochemical characterisation, including in vitro and in vivo assays, for a proteinase inhibitor purified from Clitoria fairchildiana seeds (CFPI). Purification was performed by hydrophobic interaction and gel filtration chromatography. Kinetic studies of the purified inhibitor showed a competitive-type inhibitory activity against bovine trypsin and chymotrypsin, with an inhibition stoichiometry of 1:1 for both enzymes. The inhibition constants against trypsin and chymotrypsin were 3.3 × 10(-10) and 1.5 × 10(-10)M, respectively, displaying a tight binding property. SDS-PAGE showed that CFPI has a single polypeptide chain with an apparent molecular mass of 15 kDa under non-reducing conditions. However, MALDI-TOF analysis demonstrated a molecular mass of 7.973 kDa, suggesting that CFPI is dimeric in solution. The N-terminal sequence of CFPI showed homology with members of the Bowman-Birk inhibitor family. CFPI remained stable to progressive heating for 30 min to each temperature range of 37 up to 100 °C and CD analysis exhibited no changes in spectra at 207 nm after heating at 90 °C and subsequent cooling. Moreover, CFPI was active over a wide pH range (2-10). In contrast, reduction with DTT resulted in a loss of inhibitory activity against trypsin and chymotrypsin. CFPI also exhibited significant inhibitory activity against larval midgut trypsin enzymes from Anagasta kuehniella (76%), Diatraea saccharalis (59%) and Heliothis virescens (49%). Its insecticidal properties were further analysed by bioassays and confirmed by negative impact on A. kuehniella development.

Peptide code-on-a-microplate for protease activity analysis via MALDI-TOF mass spectrometric quantitation.

A peptide-encoded microplate was proposed for MALDI-TOF mass spectrometric (MS) analysis of protease activity. The peptide codes were designed to contain a coding region and the substrate of protease for enzymatic cleavage, respectively, and an internal standard method was proposed for the MS quantitation of the cleavage products of these peptide codes. Upon the cleavage reaction in the presence of target proteases, the coding regions were released from the microplate, which were directly quantitated by using corresponding peptides with one-amino acid difference as the internal standards. The coding region could be used as the unique "Protease ID" for the identification of corresponding protease, and the amount of the cleavage product was used for protease activity analysis. Using trypsin and chymotrypsin as the model proteases to verify the multiplex protease assay, the designed "Trypsin ID" and "Chymotrypsin ID" occurred at m/z 761.6 and 711.6. The logarithm value of the intensity ratio of "Protease ID" to internal standard was proportional to trypsin and chymotrypsin concentration in a range from 5.0 to 500 and 10 to 500 nM, respectively. The detection limits for trypsin and chymotrypsin were 2.3 and 5.2 nM, respectively. The peptide-encoded microplate showed good selectivity. This proposed method provided a powerful tool for convenient identification and activity analysis of multiplex proteases.

A chymotrypsin from the Digestive Tract of California Spiny Lobster, Panulirus interruptus: Purification and Biochemical Characterization.

A chymotrypsin was purified from the gastric juice of California spiny lobster (Panulirus interrutpus), using preparative electrophoresis and affinity chromatography on agarose-p-aminobenzamidine. The molecular mass was estimated by polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions to be 28 kDa. Chymotrypsin activity was totally inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin. Lobster chymotrypsin had optimal pH 7.0-8.0 and temperature of 55 °C. The enzyme is highly stable under a wide range of pH (retaining up to 80 % of activity after 1 h of incubation at pH 3.0, 5.0, and 12.0), showing higher stability at pH 8.0, and was inactivated after 20 min at 55 °C. Lobster chymotrypsin was able to hydrolyze protein substrates at as low as pH 3.0. These results are consistent with the findings of enzyme stability. Activity was assessed after incubation of enzyme with different organic solvents (in the range of 10-50 %); when tested in the presence of acetone, ethanol, propanol, and butanol, lobster chymotrypsin residual activity was >80 %; whereas in the presence of dimethyl sulfoxide (DMSO) and toluene, lobster chymotrypsin residual activity was <80 %. Deduced amino acid sequence, corroborated by mass spectrometry, was determined.

American Pancreatic Association Practice Guidelines in Chronic Pancreatitis: evidence-based report on diagnostic guidelines.

The diagnosis of chronic pancreatitis remains challenging in early stages of the disease. This report defines the diagnostic criteria useful in the assessment of patients with suspected and established chronic pancreatitis. All current diagnostic procedures are reviewed, and evidence-based statements are provided about their utility and limitations. Diagnostic criteria for chronic pancreatitis are classified as definitive, probable, or insufficient evidence. A diagnostic (STEP-wise; survey, tomography, endoscopy, and pancreas function testing) algorithm is proposed that proceeds from a noninvasive to a more invasive approach. This algorithm maximizes specificity (low false-positive rate) in subjects with chronic abdominal pain and equivocal imaging changes. Furthermore, a nomenclature is suggested to further characterize patients with established chronic pancreatitis based on TIGAR-O (toxic, idiopathic, genetic, autoimmune, recurrent, and obstructive) etiology, gland morphology (Cambridge criteria), and physiologic state (exocrine, endocrine function) for uniformity across future multicenter research collaborations. This guideline will serve as a baseline manuscript that will be modified as new evidence becomes available and our knowledge of chronic pancreatitis improves.

Oligopeptide-decorated liquid crystal droplets for detecting proteases.

We prepared the bioactive liquid crystal (LC) droplets by decorating an oligopeptide at the LC/aqueous interface to stabilize the LC droplets. These LC droplets can be used to detect proteases through the specific cleavage of protease on an oligopeptide leading to a bipolar-to-radial transition of LC configuration inside the droplets.

Multiplexed electrochemical detection of trypsin and chymotrypsin based on distinguishable signal nanoprobes.

In this work, we developed a novel multisignal output for simultaneous detection of multiple proteases by using nanoprobes labeled with distinguishable electrochemical probes. First, biotinylated peptide1 (S1) and biotinylated peptide2 (S2) were associated with biotinylated DNA1 and DNA2 via biotin-streptavidin interaction, forming DNA1-S1 and DNA2-S2, respectively. Two distinguishable signal nanoprobes (DNA1'-Au NPs-Thi and DNA2'-Au NPs-Fc) were prepared by initial assembling DNA1' and DNA2' on the Au NPs surface, respectively, and then carrying corresponding thionine (Thi) and 6-(Ferrocenyl)hexanethiol (Fc). Then, the peptide substrates (DNA1-S1 and DNA2-S2) were immobilized on gold electrode surface through Au-S bonds, and the DNA1'-Au NPs-Thi and DNA2'-Au NPs-Fc were assembled to the peptide-DNA-modified electrode surface via DNA hybridization. The targets of trypsin and chymotrypsin can specifically recognize and cleave peptides with different sequences, releasing DNA1'-Au NPs-Thi and DNA2'-Au NPs-Fc from the electrode surface into solution, thus decreasing the current of Thi and Fc. The decrease in the electrochemical currents of the two signal nanoprobes enables us to simultaneously and quantitatively determine the targets trypsin and chymotrypsin. More importantly, this strategy can be extended easily by designing various proteases-specific peptide substrates and utilizing corresponding electrochemical detectable elements for simultaneous multiplex protease assay in various biosystems.

Assessment of proteasome concentration and chymotrypsin-like activity in plasma of patients with newly diagnosed multiple myeloma.

The ubiquitin-proteasome pathway is implicated in the pathogenesis of many haematologic malignancies, including multiple myeloma. Under conditions of rapid cell turnover and growth rate, proteasomes are returned into circulation. The measurement of their levels or activity could offer a new approach to diagnosis, prognosis and monitoring of anticancer treatment in carcinoma patients. We analysed proteasome concentration and chymotrypsin-like (ChT-L) activity in the plasma of 64 patients with a newly diagnosed multiple myeloma and 30 healthy volunteers. The values were found to be significantly higher in the studied patients and advanced disease stages compared to the control group, and decreased significant after chemotherapy. Both proteasome concentration and ChT-L activity correlated with adverse prognostic factors, such as lactate dehydrogenase and ß2-macroglobulin. We also showed that proteasome concentration positively correlates with IL-6 level, as opposed to proteasome ChT-L activity. Of note, higher proteasome ChT-L activity, unlike the concentration, was proved to be an indicator of a shorter progression free survival, constituting thereby an important prognostic marker.

Carbon nanotube-enhanced polarization of fluorescent peptides: a novel amplification strategy for homogeneous detection of proteases.

A new fluorescence polarization (FP) amplification strategy based on the use of multiwalled carbon nanotubes (MWCNTs) as the FP enhancer was developed for the simple, sensitive, and universal monitoring of protease activity in homogeneous solution. A fluorophore-labeled peptide that includes a protease-cleavable element and ten histidine residues for binding MWCNTs is adsorbed on MWCNTs through strong π-π stacking and electrostatic interactions. When the fluorophore-labeled peptide/MWCNT complexes are exposed to a protease target, specific peptide cleavage by the protease target occurs, thus releasing fragments carrying the fluorophore from the surface of MWCNTs, which in turn results in a significant decrease in the FP value. The detection limits of this assay for two proteases, thrombin and chymotrypsin (CTP), were estimated to be 0.5 pM and 0.3 pM, respectively. In addition, it is also demonstrated that this MWCNT-enhanced FP assay is suitable for protease inhibitor screening.