Activity-based enrichment of matrix metalloproteinases using reversible inhibitors as affinity ligands

Matrix metalloproteinases (MMPs) are zinc dependent metalloproteases characterized by the ability to cleave extracellular matrix and many other extracellular proteins. MMP activity is tightly regulated but disturbances in this regulation can contribute to various disease processes characterized by a progressive destruction of the extracellular matrix. The ability to profile classes of enzymes based on functionally related activities would greatly facilitate research about the involvement of MMPs in physiological and/or pathological states. Here we describe the characterization of an affinity sorbent using an immobilized reversible inhibitor as a stationary phase for the activity-based enrichment of MMPs from biological samples. With a ligand density of 9.8 mM and binding constant of 58 mumol/l towards MMP-12, the capturing power of the affinity sorbent was strong enough to extract MMP-12 spiked into serum with high selectivity from relatively large sample volumes. Experiments with endogenous inhibitors revealed that MMP-12 extraction is strictly activity-dependent, offering powerful means to monitor NIMP activities in relation to physiological and/or pathological events by using affinity extraction as a first step in an MMP profiling method. (C) 2003 Elsevier B.V. All rights reserved

Pyrococcus furiosus glyceraldehyde-3-phosphate oxidoreductase has comparable W6+/5+ and W5+/4+ reduction potentials and unusual [4Fe-4S] EPR properties

AbstractPyrococcus furiosus glyceraldehyde 3-phosphate oxidoreductase has been characterized using EPR-monitored redox titrations. Two different W signals were found. W15+ is an intermediate species in the catalytic cycle, with the midpoint potentials Em(W6+/5+)=−507 mV and Em(W5+/4+)=−491 mV. W25+ represents an inactivated species with Em(W6+/5+)=−329 mV. The cubane cluster exhibits both S=3/2 and S=1/2 signals with the same midpoint potential: Em([4Fe-4S]2+/1+)=−335 mV. The S=1/2 EPR signal is unusual with all g values below 2.0. The titration results combined with catalytic voltammetry data are consistent with electron transfer from glyceraldehyde 3-phosphate first to the tungsten center, then to the cubane cluster and finally to the ferredoxin

Activity-based matrix metallo-protease enrichment using automated, inhibitor affinity extractions

An automated inhibitor affinity extraction method for the activity-based enrichment of matrix metalloproteases (MMPs) is presented. Samples containing purified MMP-12 were first extracted at different flow rates in a syringe pump setup, using cartridges packed with an MMP inhibitor affinity sorbent based on an immobilized hydroxamic acid containing peptide (PLG-NHOH) with mu mol/L MMP affinity. Faster extractions, a reduced number of manual manipulations, and higher extraction yields (98.9%-99.3%) were obtained over the whole flow rate range compared to batch extractions. Application of the method to synovial fluid from a rheumatoid arthritis patient followed by gelatin-zymography revealed a strong enrichment of distinct MMPs from this biological sample that were not clearly visible in the original sample. The use of an auto-sampler and a solid-phase extraction (SPE) workstation allowed full automation of the extraction procedure with the potential for on-line coupling to further sample preparation and analytical steps. MMP-12 extractions were optimized showing that ligand density is an important factor with a clear extraction yield optimum around 5 to 7.5 mmol/L. Conditioning of the stationary phase for 1 week prior to use resulted in a further slight increase in extraction yield. Under optimal conditions, an extraction yield of 99.5% was reached with a cartridge contact time of only 13 s for MMP-12. The efficacy of the extraction method for activity-based MMP profiling was further improved by the use of a broad-spectrum MMP inhibitor with nmol/L affinity (TAPI-2). This resulted in an increased extraction yield for all tested MMPs. For MMP-1, -7, -8, -10, -12, and -13 extraction yields of at least 98.8% were obtained, while for MMP-9 (full length and catalytic domain) an extraction yield of at least 96.1% was reached

Chemically modified, immobilized trypsin reactor with improved digestion efficiency

Tryptic digestion followed by identification using mass spectrometry is an important step in many proteomic studies. Here, we describe the preparation of immobilized, acetylated trypsin for enhanced digestion efficacy in integrated protein analysis platforms. Complete digestion of cytochrome c was obtained with two types of modified-trypsin beads with a contact time of only 4 s, while corresponding unmodified-trypsin beads gave only incomplete digestion. The digestion rate of myoglobin, a protein known to be rather resistant to proteolysis, was not altered by acetylating trypsin and required a buffer containing 35% acetonitrile to obtain complete digestion. The use of acetylated-trypsin beads led to fewer interfering tryptic autolysis products, indicating an increased stability of this modified enzyme. Importantly, the modification did not affect trypsin's substrate specificity, as the peptide map of myoglobin was not altered upon acetylation of immobilized trypsin. Kinetic digestion experiments in solution with low-molecular-weight substrates and cytochrome c confirmed the increased catalytic efficiency (lower K-M and higher k(cat)) and increased resistance to autolysis of trypsin upon acetylation. Enhancement of catalytic efficiency was correlated with the number of acetylations per molecule. The favorable properties of the new chemically modified trypsin reactor should make it a valuable tool in automated protein analysis systems

Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua

An important group of antimalarial drugs consists of the endoperoxide sesquiterpene lactone artemisinin and its derivatives. Only little is known about the biosynthesis of artemisinin in Artemisia annua L., particularly about the early enzymatic steps between amorpha-4,11-diene and dihydroartemisinic acid. Analyses of the terpenoids from A. annua leaves and gland secretory cells revealed the presence of the oxygenated amorpha-4,11-diene derivatives artemisinic alcohol, dihydroartemisinic alcohol, artemisinic aldehyde, dihydroartemisinic aldehyde and dihydroartemisinic acid. We also demonstrated the presence of a number of biosynthetic enzymes such as the amorpha-4,11-diene synthase and the - so far unknown - amorpha-4,11-diene hydroxylase as well as artemisinic alcohol and dihydroartemisinic aldehyde dehydrogenase activities in both leaves and glandular trichomes. From these results, we hypothesise that the early steps in artemisinin biosynthesis involve amorpha-4,11-diene hydroxylation to artemisinic alcohol, followed by oxidation to artemisinic aldehyde, reduction of the C11-C13 double bond to dihydroartemisinic aldehyde and oxidation to dihydroartemisinic aci