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Botulinum neurotoxins (BoNTs) are a family of protein toxins consisting of seven known serotypes (BoNT/A—BoNT/G) and multiple subtypes within the serotypes, and all of which cause the disease botulism—a disease of great public health concern. Accurate detection of BoNTs in human clinical samples is therefore an important public health goal. To achieve this goal, our laboratory developed a mass spectrometry-based assay detecting the presence of BoNT via its enzymatic activity on a peptide substrate. Recently, publications reported the use of new peptide substrates to detect BoNT/A and /B with improved results over other peptide substrates. However, the authors did not provide results of their peptide substrate on multiple subtypes of BoNT. In this work, we describe the results of testing the new substrates with multiple BoNT/A and /B subtypes and find that the substrates cannot detect many subtypes of BoNT/A and /B.

Human pluripotent stem cell derived neural progenitor cells (hNPCs) have the unique properties of long-term in vitro expansion as well as differentiation into the various neurons and supporting cell types of the central nervous system (CNS). Because of these characteristics, hNPCs have tremendous potential in the modeling and treatment of various CNS diseases and disorders. However, expansion and neuronal differentiation of hNPCs in quantities necessary for these applications is not possible with current two dimensional (2-D) approaches. Here, we used a fully defined peptide substrate as the basis for a microcarrier (MC)-based suspension culture system. Several independently derived hNPC lines were cultured on MCs for multiple passages as well as efficiently differentiated to neurons. Finally, this MC-based system was used in conjunction with a low shear rotating wall vessel (RWV) bioreactor for the integrated, large-scale expansion and neuronal differentiation of hNPCs. Overall, this fully defined and scalable biomanufacturing system will facilitate the generation of hNPCs and their neuronal derivatives in quantities necessary for basic and translational applications. Statement of Significance In this work, we developed a microcarrier (MC)-based culture system that allows for the expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells (hNPCs) under defined conditions. In turn, this MC approach was implemented in a rotating wall vessel (RWV) bioreactor for the large-scale expansion and neuronal differentiation of hNPCs. This work is of significance as it overcomes current limitations of conventional two dimensional (2-D) culture systems to enable the generation of hNPCs and their neuronal derivatives in quantities required for downstream applications in disease modeling, drug screening, and regenerative medicine.

Protein quality control depends on the tight regulation of interactions between molecular chaperones and polypeptide substrates. Substrate release from the chaperone Hsp70 is triggered by nucleotide-exchange factors (NEFs) that control folding and degradation fates via poorly understood mechanisms. We found that the armadillo-type NEFs budding yeast Fes1 and its human homolog HspBP1 employ flexible N-terminal release domains (RDs) with substrate-mimicking properties to ensure the efficient release of persistent substrates from Hsp70. The RD contacts the substrate-binding domain of the chaperone, competes with peptide substrate for binding and is essential for proper function in yeast and mammalian cells. Thus, the armadillo domain engages Hsp70 to trigger nucleotide exchange, whereas the RD safeguards the release of substrates. Our findings provide fundamental mechanistic insight into the functional specialization of Hsp70 NEFs and have implications for the understanding of proteostasis-related disorders, including Marinesco–Sjogren syndrome. Nucleotide exchange factors (NEFs) trigger substrate release from molecular chaperone Hsp70. The authors found that armadillo-type NEFs (yeast Fes1, human HspBP1) competitively prevent rebinding of released substrate.

The β-secretase (BACE1) initiates the generation of toxic amyloid-β peptide (Aβ) from amyloid-β precursor protein (APP), which was widely considered to play a key role in the pathogenesis of Alzheimer’s disease (AD). Here, a novel microfluidics-based mobility shift assay (MMSA) was developed, validated, and applied for the screening of BACE1 inhibitors for AD. First, the BACE1 activity assay was established with a new fluorescent peptide substrate (FAM-EVNLDAEF) derived from the Swedish mutant APP, and high-quality ratiometric data were generated in both endpoint and kinetic modes by electrophoretic separation of peptide substrate from the BACE1 cleaved product (FAM-EVNL) before fluorescence quantification. To validate the assay, the inhibition and kinetic parameter values of two known inhibitors (AZD3839 and AZD3293) were evaluated, and the results were in good agreement with those reported by other methods. Finally, the assay was applied to screen for new inhibitors from a 900-compound library in a 384-well format, and one novel hit (IC50 = 26.5 ± 1.5 μM) was identified. Compared with the common fluorescence-based assays, the primary advantage of the direct MMSA was to discover novel BACE1 inhibitors with lower auto-fluorescence interference, and its superb capability for kinetic study.

Botulinum neurotoxins (BoNTs) are the most toxic proteins in nature. Endopeptidase-mass-spectrometry (Endopep-MS) is used as a specific and rapid in-vitro assay to detect BoNTs. In this assay, immunocaptured toxin cleaves a serotype-specific-peptide-substrate, and the cleavage products are then detected by MS. Here we describe the design of a new peptide substrate for improved detection of BoNT type A (BoNT/A). Our strategy was based on reported BoNT/A-SNAP-25 interactions integrated with analysis method efficiency considerations. Integration of the newly designed substrate led to a 10-fold increase in the assay sensitivity both in buffer and in clinically relevant samples.

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.

Lon is a widely-conserved housekeeping protease found in all domains of life. Bacterial Lon is involved in the recovery from various types of stress, including tolerance to fluoroquinolone antibiotics, and is linked to pathogenesis in a number of organisms. However, detailed functional studies of Lon have been limited by the lack of selective, cell-permeable inhibitors. Here we describe the use of positional scanning libraries of hybrid peptide substrates to profile the primary sequence specificity of bacterial Lon. In addition to identifying optimal natural amino acid binding preferences, we identified several non-natural residues that were leveraged to develop optimal peptide substrates as well as a potent peptidic boronic acid inhibitor of Lon. Treatment ofE. coliwith this inhibitor promotes UV-induced filamentation and reduces tolerance to ciprofloxacin, phenocopying establishedlon-deletion phenotypes. It is also non-toxic to mammalian cells due to its increased selectivity for Lon over the proteasome. Our results provide new insight into the primary substrate specificity of Lon and identify substrates and an inhibitor that will serve as useful tools for dissecting the diverse cellular functions of Lon.

The kinetic chromogenic endotoxin assay measures the release of p-nitroaniline from the chromogenic peptide substrate Ac-IEAR-pNA. As part of our project to purify large quantities of human butyrylcholinesterase (HuBChE), we evaluated pure HuBChE for endotoxin levels. We found that HuBChE contributed up to 90% of the yellow p-nitroaniline product in a standard endotoxin assay through the catalytic hydrolysis of Ac-IEAR-pNA with a rate constant of 0.016 min(-1) and a Km of 2.9 mM in potassium phosphate buffer pH 7.0 at 24 °C. Thus, endotoxin concentrations for native BChE are artificially high in the kinetic chromogenic assay. Destruction of HuBChE catalytic activity by boiling yields endotoxin concentrations that more accurately reflect the endotoxin concentration in purified HuBChE preparations.

Clostridium botulinum produces botulinum neurotoxins (BoNTs) that are one of the most poisonous substances. In order to respond to public health emergencies, there is a need to develop sensitive and specific methods for detecting botulinum toxin in various clinical matrices. Our laboratory has developed a mass spectrometry-based Endopep-MS assay that is able to rapidly detect and differentiate BoNT serotypes A–G by immunoaffinity capture of toxins and detection of unique cleavage products of peptide substrates. To improve the sensitivity of the Endopep-MS assay for the detection of BoNT serotype G, we report here the optimization of synthetic peptide substrates through systematic substitution, deletion, and incorporation of unnatural amino acids. Our data show that the resulting optimized peptides produced a significant improvement (two orders of magnitude) in assay sensitivity and allowed the detection of 0.01 mouseLD50 toxin present in buffer solution.

Graphical abstract, Phosphotyrosine peptides are useful starting points for inhibitor design and for the search for protein tyrosine phosphatase (PTP) phosphoprotein substrates. To identify novel phosphopeptide substrates of PTP1B, we developed a computational prediction protocol based on a virtual library of protein sequences with known phosphotyrosine sites. To these we applied sequence-based methods, biologically meaningful filters and molecular docking. Five peptides were selected for biochemical testing of their potential as PTP1B substrates. All five peptides were equally good substrates for PTP1B compared to a known peptide substrate whereas appropriate control peptides were not recognized, showing that our protocol can be used to identify novel peptide substrates of PTP1B.

With increasing emergence of multi-drug resistant infections, there is a dire need for new classes of compounds that act through unique mechanisms. In this work, we describe the discovery and optimization of a novel series of inhibitors of bacterial methionine aminopeptidase (MAP). Through a high-throughput screening campaign, one azepinone amide hit was found that resembled the native peptide substrate and possessed moderate biochemical potency against three bacterial isozymes. X-ray crystallography was used in combination with substrate-based design to direct the rational optimization of analogs with sub-micromolar potency. The novel compounds presented here represent potent broad-spectrum biochemical inhibitors of bacterial MAP and have the potential to lead to the development of new medicines to combat serious multi-drug resistant infections.

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous substances known to humankind. It is essential to have a simple, quick, and sensitive method for the detection and quantification of botulinum toxin in various media, including complex biological matrices. Our laboratory has developed a mass spectrometry-based Endopep–MS assay that is able to rapidly detect and differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique cleavage products of peptide substrates. Botulinum neurotoxin type E (BoNT/E) is a member of a family of seven distinctive BoNT serotypes (A–G) and is the causative agent of botulism in both humans and animals. To improve the sensitivity of the Endopep–MS assay, we report here the development of novel peptide substrates for the detection of BoNT/E activity through systematic and comprehensive approaches. Our data demonstrate that several optimal peptides could accomplish 500-fold improvement in sensitivity compared with the current substrate for the detection of both not-trypsin-activated and trypsin-activated BoNT/E toxin complexes. A limit of detection of 0.1 mouse LD 50 /ml was achieved using the novel peptide substrate in the assay to detect not-trypsin-activated BoNT/E complex spiked in serum, stool, and food samples.

Botulinum neurotoxins (BoNTs) are bacterial proteins that cause botulism, a life-threatening disease. The Endopep-MS assay permits rapid detection and serotypic differential diagnosis of BoNTs. The serotype-specific nature of this assay requires that each serum sample be aliquoted and individually tested, which in addition to the limited volume of clinical samples, especially in infants, points to the need for a multiplex assay. However, previous attempts to develop such an assay have been challenging, mainly due to inhibition of BoNT/A activity by the BoNT/E peptide substrate. BoNT/A and BoNT/E share the same native target protein as their substrate. We hypothesized that the steric interference between the BoNT/A and BoNT/E substrate peptides is responsible for the difficulty in simultaneously assaying these two toxins. To explore the basis for steric interference, we used the reported structure of BoNT/A in complex with SNAP-25 and modelled the structure of BoNT/E with SNAP-25. Following this thorough structural analysis, we designed a new peptide substrate for BoNT/A that maintained the assay sensitivity and allowed, for the first time, simultaneous detection of the three most abundant human botulinum serotypes. Adopting the multiplex assay will minimize the required sample volume and assay time for botulinum detection while maintaining the superior Endopep-MS assay performance.

An optimized peptide substrate was used to measure protein kinase B (PKB) activity in single cells. The peptide substrate was introduced into single cells, and capillary electrophoresis was used to separate and quantify nonphosphorylated and phosphorylated peptide. The system was validated in three model pancreatic cancer cell lines before being applied to primary cells from human pancreatic adenocarcinomas propagated in nude mice. As measured by phosphorylation of peptide substrate, each tumor cell line exhibited statistically different median levels of PKB activity (65%, 21%, and 4% phosphorylation in PANC-1 (human pancreatic carcinoma), CFPAC-1 (human metastatic ductal pancreatic adenocarcinoma), and HPAF-II cells (human pancreatic adenocarcinoma), respectively) with CFPAC-1 cells demonstrating two populations of cells or bimodal behavior in PKB activation levels. The primary cells exhibited highly variable PKB activity at the single cell level, with some cells displaying little to no activity and others possessing very high levels of activity. This system also enabled simultaneous characterization of peptidase action in single cells by measuring the amount of cleaved peptide substrate in each cell. The tumor cell lines displayed degradation rates statistically similar to one another (0.02, 0.06, and 0.1 zmol pg(-1) s(-1), for PANC-1, CFPAC-1, and HPAF-II cells, respectively) while the degradation rate in primary cells was 10-fold slower. The peptide cleavage sites also varied between tissue-cultured and primary cells, with 5- and 8-residue fragments formed in tumor cell lines and only the 8-residue fragment formed in primary cells. These results demonstrate the ability of chemical cytometry to identify important differences in enzymatic behavior between primary cells and tissue-cultured cell lines.

Proteases are essential for the proliferation and growth of bacteria, and are also known to contribute to bacterial virulence. This makes them interesting candidates as diagnostic and therapeutic targets for infectious diseases. In this review, the authors discuss the most recent developments and potential applications for bacterial proteases in the diagnosis and treatment of bacterial infections. Current and future bacterial protease targets are described and their limitations outlined.

DNA-PK/Gene expression/Hypoxia/Reoxygenation. In order to elucidate the mechanism of concerted expression of various proteins related to DNA-PK activity, we ex amined the relationship of the expression of these proteins with that of a transcription factor Sp1. We also explored whether the expression of genes related to DNA-PK activity and Sp1 changed after hypoxia and reoxygenation. RT-PCR was employed to measure expression of various proteins related to DNA-PK activity in peripheral blood cells (PBLs) of normal healthy volunteers and cancer patients. M059K and DLD1 cells were made hypoxic with the hypoxia chamber, and the expression of various proteins in the cells was examined. DNA-PK activity was measu red using synthetic peptide substrate mimicking the sequence around Ser15 of human p53. Expressi on of examined genes whose expression was related to DNA-PK activity was significantly correlated with expression of Sp1. The expression of most genes lost its relationship with the expression of Sp1 after hypoxic exposure and reoxygenation. DNA-PK activity tended to decrease after hypoxic exposure and to recover after reoxygenation. Sp1 may control expression of genes related to DNA-PK activity. Expression of those genes deviated from the control Sp1 under hypoxia and reoxygenation. The control mechanism for expression of genes related to DNA-PK activity under hypoxia and reoxygenation may be different from that under aerobic conditions.

The acronym CK2 denotes a highly pleiotropic Ser/Thr protein kinase whose over-expression correlates with neoplastic growth. A vexed question about the enigmatic regulation of CK2 concerns the actual existence in living cells of the catalytic (alpha and/or alpha') and regulatory beta-subunits of CK2 not assembled into the regular heterotetrameric holoenzyme. Here we take advantage of novel reagents, namely a peptide substrate and an inhibitor which discriminate between the holoenzyme and the catalytic subunits, to show that CK2 activity in CHO cells is entirely accounted for by the holoenzyme. Transfection with individual subunits moreover does not give rise to holoenzyme formation unless the catalytic and regulatory subunits are co-transfected together, arguing against the existence of free subunits in CHO cells.

A new label-free method for the detection of apoptosis was proposed based on colorimetric assay of caspase-3 activity using an unlabeled Asp-Glu-Val-Asp (DEVD)-containing peptide substrate and unmodified gold nanoparticles (AuNPs).

High-throughput screening in the 1536-well format has been largely restricted to solution-based and cell-based screens. In this article, we show the feasibility of a completely automated, robust scintillation proximity assay in the 1536-well format that is suitable to identify inhibitors for a serine/threonine kinase from a compound library. The introduction of [(33)P]phosphate into a biotinylated peptide substrate mirrors the activity of the kinase. The peptide is immobilized on streptavidin-coated LEADseeker imaging beads and [(33)P]phosphate incorporation is detected with the LEADseeker imaging system of Amersham Pharmacia Biotech. To improve the liquid handling procedures for imaging bead suspensions in the low microliter range, we developed a novel trough with an integrated stirring function. A comparison of the 1536-well assay to a 384-well assay revealed a comparable assay quality with Z' factors of about 0.7 for the 384-well format and 0.6 for the 1536-well format. In an automated screen of a random compound collection, 94.4% of the inhibitory compounds could be identified with both assay formats. Dose-response curves were performed for a selection of identified kinase inhibitors and revealed similar IC(50) values for both assay formats.

The West Nile Virus (WNV) has been a worldwide epidemic since the early 1990s. Currently there are no therapeutic treatments for WNV infections. One particular avenue of treatment is inhibition of the NS2B-NS3 protease, an enzyme that is crucial for WNV replication. In our effort to increase the number of NS2B-NS3 protease inhibitors, we report a novel FRET-based high throughput assay for the discovery of WNV NS2B-NS3 protease inhibitors. For this assay, a FRET-based peptide substrate was synthesized and kinetically characterized with the NS2B-NS3 protease. The new substrate exhibits a K(m) of 3.35 ± 0.31 μM, a k(cat) of 0.0717 ± 0.0016 s(-1) and a k(cat)/K(m) of 21,400 ± 2000 M(-1) s(-1).

The design and synthesis of hydroxyethylamine isosteres as inhibitors of cathepsin D based on SAR data have been accomplished. A library of 96 of these hydroxyethylamine isosteres are described and many have proven to be very potent inhibitors of human cathepsin D activity as measured using a fluorometric assay technique, via peptide substrate Ac-Glu-Glu(Edans)-Lys-Pro-Ile-Cys-Phe-Phe-Arg-Leu-Gly-Lys(Methyl Red)-Glu-NH(2). Compounds showing strongest inhibition of cathepsin D activity were those that contain a hydroxyethyl-N'-2- or N'-(4-chlorophenyl)piperazine moiety (IC(50) values range from 0.55 to 8.5 nM), with N'-(2-pyrimidyl)piperizine (IC(50) values range from 0.5 to 21.6 nM), with N-N'- L-piperazinocolinamide (IC(50) values range from 0.001 - 0.25 nM), or N-N'-L-piperazinocolin-N-methylamide (IC(50) values range from 0.015 - 7.3 nM).

Patients with chronic obstructive pulmonary disease (COPD) are prone to acute exacerbations associated with increased morbidity and mortality. One potential group of enzymes causing tissue destruction in this disease includes neutrophil proteinase elastase (NE), collagenase-2 (matrix metalloproteinase-8 (MMP-8)) and gelatinase B (MMP-9). We investigated the activity of NE and the levels of MMP-8 and MMP-9 in a longitudinal setting at and after COPD exacerbation using a non-invasive technique, i.e., induced sputum, to ascertain whether these proteinases play a role in COPD exacerbation.The study included healthy non-smokers (n=32), healthy smokers (n=28), patients with stable COPD (n=15), COPD patients with acute exacerbations (exa) (n=10) and their recovery (n=8) after 4 weeks. NE activity by synthetic peptide substrate and spectrophotometry, MMP-8 levels by immunofluorometry and MMP-9 levels by ELISA were analysed from induced sputum supernatants.NE activity and the level of MMP-8 increased highly significantly in patients with COPD exacerbation compared to stable COPD and controls (NE: p = 0.001 and p0.0001; MMP-8: p0.001 and p0.0001). Paired samples showed a decrease of these proteinases during the recovery period after exacerbation (p = 0.03, p = 0.04). The proteinase levels correlated not only with the percentage and number of neutrophils but also with the lung function parameters (FEV1/FVC and diffusion capacity).COPD exacerbations are associated with neutrophil recruitment into the airways but also transient activation and/or elevation of tissue destruction proteinases, such as NE and MMP-8, which can be detected from the induced sputum supernatants of these COPD patients.

To develop a novel cerebrospinal fluid (CSF) beta-secretase-1 activity assay and evaluate beta-secretase-1 (BACE-1) activity as a potential biomarker in human Alzheimer's disease.The assay consisted of an enzymatic reaction of CSF samples with an optimized beta-secretase peptide substrate and the cleavage products were detected using a neo-epitope specific antibody.The CSF BACE-1 activity assay described exhibits time, temperature, dose, and pH dependence, with sensitivity down to1 pM of recombinant BACE-1 enzyme, and is completely blocked by BACE-1 inhibitors. The endogenous BACE-1 enzyme in CSF appears to exist as a c-terminally truncated protein, based on both western blotting and capture-based activity assays. In a small cohort of human subjects, an age-dependent increase in CSF BACE activity was observed (~1.0 pM/year, p0.05). In Alzheimer's disease subjects, a significant decline in age-adjusted CSF BACE activity was observed compared to controls (56% in the log-transformed scale, p=0.02).We have developed a robust assay to measure CSF BACE-1 activity which could serve as a potential biomarker in human Alzheimer's disease subjects.

A new chromogenic peptide substrate assay kit was used to measure factor XII (FXII) levels in plasma samples from 115 male patients with heart disease awaiting cardiac surgery, 40 age-matched normal healthy male blood donors and 20 patients before, during and after cardiopulmonary bypass surgery (CPB). Kallikrein-like and FXIIa-like activities were also determined in the CPB patient group. FXII levels were significantly lower (p = 0.0049) in the heart disease patients awaiting surgery when compared with values for the healthy donors and 13 patients (11.3%) had FXII levels below 50% compared with 1 normal donor (2.5%). During CPB significant decreases in FXII levels and significant increases in FXIIa-like and kallikrein-like activities were found indicating activation of the FXII-plasma kallikrein pathway during CPB.

The assay is based on a modified pro-urokinase in which the activation sequence, normally recognised by plasmin was replaced by a sequence that is specifically recognised by MMPs (figure 1). A chromogenic peptide substrate for urokinase is then used to measure the active urokinase, generated through MMP activation of the modified urokinase. The assay uses an MMP-2 specific antibody, coated 96-well microtitre plate to confer MMP-2 specificity. MMP-2 is captured from biological fluids or standards (proMMP-2). Immobilised latent MMP-2 is then activated using APMA to measure total MMP-2. Differential activation enables the analysis of the activity of both the already active and latent forms of MMP-2.

We have developed an automated chromogenic peptide substrate assay for factor XII (FXIIcs) on a Cobas Mira S Plus clinical chemistry analyser using a new commercially available kit. This was used to determine factor XII (FXII) levels in plasma samples from 320 blood donors, 206 patients with a history of venous thrombosis and 74 lupus anticoagulant positive (LA+) patients. Results were compared with those obtained in a clotting assay for FXII (FXIIct) and an immunochemical assay (FXIIag). A satisfactory correlation coefficient of 0.92 and a regression line equation of y = 7.898 + 0.871x was obtained between FXIIcs and FXIIct in the 320 blood donors. Levels of FXII below the calculated normal range were found in nine blood donors (2.8%) and 16 venous thrombosis patients (7.8%). The blood donors and patients with venous thrombosis with low FXIIcs values had FXII levels below our lower limits of normal for both FXIIct and FXIIag; all were lupus anticoagulant negative. When FXII levels were determined in the 74 LA+ patients, 27 (36.5%) gave markedly lower FXII values in the FXIIct when compared with the FXIIcs. FXIIag levels corresponded with FXIIcs. The automated FXIIcs assay is therefore lupus anticoagulant insensitive and allows us to measure FXII levels accurately and routinely in large numbers of patient samples.

An approach utilizing robotics (automation) for the rapid and reliable determination of protease inhibitor concentration in rat plasma samples is described. The bioassay protocol using an immobilized peptide substrate allows high sample throughput, compatible with parallel synthesis/SAR development strategy.

A process for semicontinuous production of matrilysin zymogen secreted from recombinant Chinese hamster ovary (CHO) cells was developed. The zymogen was purified to apparent homogeneity by sequential ion-exchange and metal chelation chromatography. These processes were scaled-up to purify gram quantities of the zymogen. The N-terminus of the secreted zymogen from the recombinant cells was the same as the observed sequence of the zymogen from natural sources. Furthermore, activation and autocatalysis of the recombinant zymogen resulted in a form with an N-terminus identical to that of the corresponding native enzyme. The three C-terminal amino acids of both the recombinant zymogen and the corresponding smaller activated form were missing. Activated matrilysin was shown to have activity against a synthetic peptide substrate. The large quantities of matrilysin that can be produced and purified from the recombinant CHO cells will be useful in determination of the structure of matrilysin.

Chromogenic peptide substrates were first introduced into research laboratories in the early 1970s and were quickly utilised to develop assays for the determination of enzymes, proenzymes and inhibitors of the coagulation system. These assays were gradually introduced into coagulation and clinical chemistry laboratories as laboratory tools in the diagnosis and treatment of coagulation disorders. From the knowledge of the structures of the natural substrates attacked by enzymes other than those of the coagulation system or by synthesis and random screening, substrates for enzymes of the fibrinolytic, plasma and glandular kallikrein and complement systems were produced. These allowed various research groups to develop assays for components of these systems and subsequently led to the use of these assays in studies on various clinical conditions. Substrates for activated protein C ensured that assays for this enzyme and its inhibitors could be developed and introduced into the haematological routine. With the introduction of substrates for limulus lysate not only were assays for endotoxins in clinical samples produced but the control of all disposable products and injectables for endotoxin contamination can now be effected. Initially high costs and time-consuming manual assays were a hinderence to the general acceptance of the use of chromogenic peptide substrate assays and they were only used routinely in a few specialised laboratories. With the introduction of automated and microtitre plate methods however, these assays are are now available in most hospital laboratories. Since the first chromogenic peptide substrate was described thousands of articles have been published on the use of chromogenic substrate assays to measure proenzymes, enzyme activators, enzyme cofactors and inhibitors in blood and other body fluids in normal subjects and clinical material. We have endeavoured to cover as many of these as possible in this review.

A heparin assay method based on thrombin cleavage of a fluorogenic synthetic peptide has been evaluated in normal subjects, in patients with chronic renal disease on hemodialysis, and in patients with other disorders. The method has satisfactory accuracy, sensitivity, and reproducibility. In contrast to methods commonly used to monitor heparin therapy, it measures heparin directly and is uninfluenced by patient variables such as low antithrombin III. The method may provide valuable information in selected clinical settings such as cardiopulmonary bypass and in patients with chronic renal disease who receive heparin during hemodialysis or prior to surgery.

The heparin-accelerated neutralisation of bovine alpha and beta thrombins has been examined using a peptide substrate H-D-phenylalanyl-pipecolyl-arginine-paranitroanilide-HCl to measure thrombin amidase activity. Alpha and beta thrombins were both neutralised by antithrombin III and this neutralisation was further accelerated by the presence of small amounts of heparin. Low and high molecular weight heparin and heparins fractionated by their affinity for antithrombin III were all able to accelerate the neutralisation of alpha and beta thrombin. This work is therefore unable to confirm reports that alpha and beta thrombins have different heparin sensitivities.

Measurement of carboxypeptidase A, one of the pancreatic proteolytic enzymes, in human serum is made possible by a combination of affinity chromatography to isolate and concentrate the enzyme followed by monitoring activity spectrophotometrically with a high-turnover peptide substrate. Concentrations of enzyme in the nanogram-per-milliliter range can be determined with high precision and reliability. Initial clinical application of this method demonstrates no detectable activity in serum from normal individuals, but the enzyme is present in the sera of individuals with pancreatitis.

Components of the plasma protease systems were determined by means of chromogenic peptide substrate assays during the early stage of septicemia in 21 patients of whom 11 died. The proenzyme functional inhibition index, calculated from the measured values for plasma prekallikrein, functional kallikrein inhibition, plasminogen and functional antiplasmin and antithrombin III activities, were markedly reduced in both groups, but significantly lower in the fatal cases than in the survivors from the first day of septicemia and throughout the observation period. Fatal septicemia thus appear to be associated with a more extensive proteolytic activity in plasma than nonfatal septicemia and can be readily disclosed by calculation of the proenzyme functional inhibition index.

The kinetics of the activation of factor VIII by low concentrations of thrombin has been studied. The initial rates of increase in procoagulant activity at various concentrations of purified human factor VIII/vWFX were measured and found to obey Michaelis-Menten kinetics. The apparent Km obtained was 4.3 × 10−9 M. Purified factor VIII/vWF inhibits the reactions between thrombin and fibrinogen or chromogenic peptide substrate S-2238. Kinetic studies on the thrombin — S-2238 — factor VIII/vWF system show that factor VIII/vWF inhibits the thrombin — S-2238 reaction and an apparent Ki of 2.5 × 10−9 was obtained. Corresponding studies of factor VIII prepared by Ca2+ - mediated dissociation produced similar results, indicating that it is mainly the factor VIII part of the factor VIII/vWF complex that strongly interacts with thrombin.

Components of the coagulation and fibrinolytic systems were determined in patients undergoing open heart surgery with cardiopulmonary bypass. The variables studied included prothrombin, antithrombin-III, spontaneous plasmin activity, plasminogen and functional antiplasmin activity. The variables were measured using chromogenic peptide substrate assays. A marked, transitory, increase in spontaneous plasmin activity prior to cardiopulmonary bypass, but after heparin injection was found. A decrease in antiplasmin activity during bypass was observed, while actual plasminogen level, when correcting for hemodilution, was unchanged. Both prothrombin and antithrombin-III paralleled the decrease in hemoglobin concentration during bypass. These findings suggest that the injection of heparin induced a transient activation of the fibrinolytic system, whereas no detectable consumption of the measured coagulation variables was observed.

• Plasma prekallikrein and functional kallikrein inhibition were studied in 18 surgical patients with complicating septicemia using chromogenic peptide substrate assays. Nine patients died and nine survived. In all 18 patients plasma prekallikrein values were reduced markedly when septicemia was diagnosed. During treatment gradually increasing values were found in the survivors, whereas values remained low in the fatal cases. Significantly reduced functional plasma kallikrein inhibition was associated with the development of fatal septic shock. The findings show that determination of these components of the plasma kallikrein-kinin system gives valuable information of prognostic value in patients with septicemia. Furthermore, the chromogenic peptide substrate assays used are fast and easy to perform and therefore suitable for intensive care medicine. ( Arch Surg 1983;118:343-346)

A new chromogenic peptide substrate method, modified for assay with semi-micro tubes, Cobas Bio centrifugal analyser and microplates, was used for screening of F VIII:C in blood donors. The precision of the assays is high and the costs are reasonable. The assay with microplates is especially suitable for selection of donors for a plasma programme and for quality control in blood banks.

Using chromogenic peptide substrate assays the severity and clinical course were evaluated in 37 patients with acute pancreatitis. Retrospective clinical evaluation revealed that 20 patients had a severe disease, whereas 17 patients had mild acute pancreatitis. Seven of the patients with severe acute pancreatitis died. The proenzyme functional inhibition index (PFI index) is defined as the sum of deviations from the normal plasma pool values of plasma prekallikrein, functional kallikrein inhibition, plasminogen, antiplasmin, prothrombin and antithrombin III. Increased values are counted as positive, whereas reductions compared with the normal plasma pool values are recognized as negative. During the second day after admission the PFI index revealed significantly more negative values in severe cases than in patients with mild acute pancreatitis, -159 in severe case, -74 in mild cases (median values, P less than 0.05). The PFI index values were maintained strongly negative for the following 3 days in severe cases whereas the index was brought to positive values during the same period in patients with mild acute pancreatitis. The fatal cases revealed strongly negative PFI index values for the whole observation period. The patients with severe acute pancreatitis were earlier identified by means of the PFI index than by individual parameters also used for calculating the index. The results show that by means of the PFI index severity of acute pancreatitis can be recognized during early stages of the disease.