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Inorganic pyrophosphate (PPi) is a crucial extracellular mineralization regulator. Low plasma PPi concentrations underlie the soft tissue calcification present in several rare hereditary mineralization disorders as well as in more common conditions like chronic kidney disease and diabetes. Even though deregulated plasma PPi homeostasis is known to be linked to multiple human diseases, there is currently no reliable assay for its quantification. We here describe a PPi assay that employs the enzyme ATP sulfurylase to convert PPi into ATP. Generated ATP is subsequently quantified by firefly luciferase-based bioluminescence. An internal ATP standard was used to correct for sample-specific interference by matrix compounds on firefly luciferase activity. The assay was validated and shows excellent precision (< 3.5%) and accuracy (93-106%) of PPi spiked into human plasma samples. We found that of several anticoagulants tested only EDTA effectively blocked conversion of ATP into PPi in plasma after blood collection. Moreover, filtration over a 300,000-Da molecular weight cut-off membrane reduced variability of plasma PPi and removed ATP present in a membrane-enclosed compartment, possibly platelets. Applied to plasma samples of wild-type and Abcc6(-/-) rats, an animal model with established low circulating levels of PPi, the new assay showed lower variability than the assay that was previously in routine use in our laboratory. In conclusion, we here report a new and robust assay to determine PPi concentrations in plasma, which outperforms currently available assays because of its high sensitivity, precision, and accuracy.

Cellobiohydrolases (CBHs) in the glycoside hydrolase family 7 (GH7) (EC3.2.1.176) are the major cellulose degrading enzymes both in industrial settings and in the context of carbon cycling in nature. Small carbohydrate conjugates such as p-nitrophenyl-beta-d-cellobioside (pNPC), p-nitrophenyl-beta-d-lactoside (pNPL) and methylumbelliferyl-beta-d-cellobioside have commonly been used in colorimetric and fluorometric assays for analysing activity of these enzymes. Despite the similar nature of these compounds the kinetics of their enzymatic hydrolysis vary greatly between the different compounds as well as among different enzymes within the GH7 family. Through enzyme kinetics, crystallographic structure determination, molecular dynamics simulations, and fluorometric binding studies using the closely related compound o-nitrophenyl-beta-d-cellobioside (oNPC), in this work we examine the different hydrolysis characteristics of these compounds on two model enzymes of this class, TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium. Protein crystal structures of the E212Q mutant of TrCel7A with pNPC and pNPL, and the wildtype TrCel7A with oNPC, reveal that non-productive binding at the product site is the dominating binding mode for these compounds. Enzyme kinetics results suggest the strength of non-productive binding is a key determinant for the activity characteristics on these substrates, with PcCel7D consistently showing higher turnover rates (k(cat)) than TrCel7A, but higher Michaelis-Menten (K-M) constants as well. Furthermore, oNPC turned out to be useful as an active-site probe for fluorometric determination of the dissociation constant for cellobiose on TrCel7A but could not be utilized for the same purpose on PcCel7D, likely due to strong binding to an unknown site outside the active site.

Sugars were derivatized with N,O-dimethylhydroxylamine (DMHA) using a simple procedure. The disaccharides lactose and chitobiose and the human milk tetrasaccharides lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) were used as examples. The beta-glycosylamines were formed exclusively in good yields (80-84%). The derivatives were very well suited for RP-HPLC, giving rise to single peaks for each sugar, without the usual complications caused by mutarotation. The LNT-and LNnT-derivatives separated very well on an ordinary RP-HPLC column, despite their close structural similarity. Also, three human milk pentasaccharides (LNF I, II and III) were derivatized with DMHA. Again, good separation of these isomers was obtained. The DMHA derivatization was easily reversed. The free oligosaccharides were recovered quantitatively by mild acidic hydrolysis. To demonstrate usefulness on a preparative scale, an LNDI-rich human milk oligosaccharide fraction was derivat-ized, and three HPLC fractions (one major and two minor) were collected. Hydrolysis and desalting gave sac-charides LNDI, LNnDII, and LNDII, the latter mixed with minor amounts of LNnDI.

Leupeptin is a naturally occurring inhibitor of various proteases, in particular serine proteases. Following its discovery, the inhibitory properties of several other peptidyl argininals have been studied. The specificity of leupeptin is most likely due to the Leu-Leu-Argininal sequence, and its C-terminal aldehyde group has been suggested to enhance the binding efficiency and to be essential for function. The terminal aldehyde group makes the structure less vulnerable to carboxypeptidases. Here, we investigated whether the inhibitory function of leupeptin toward serine proteases is retained after oxidation or reduction of the aldehyde group. The oxidized form, which corresponds to the natural precursor, was shown to be superior to the reduced form in terms of inhibitory properties. However, the original leupeptin possessed enhanced inhibitory properties as compared with the oxidized form. Based on these results, new synthetic leupeptin analogues, 6-aminohexanoic acid (Ahx)-Phe-Leu-Arg-COOH and Ahx-Leu-Leu-Arg-COOH, were prepared by solid-phase peptide synthesis using the Fmoc strategy. In these analogues, the N-terminal capping acetyl group was replaced with a 6-aminohexanoyl group to allow conjugation. The structures of the modified leupeptin and the synthetic peptides were confirmed by mass spectrometry. Determination of the inhibitory properties against trypsin (IEC 3.4.21.4, Chymotrypsin IEC 3.4.21.1) revealed that these further modified tripeptides were tight binding inhibitors to their target enzyme, similar to the naturally occurring leupeptin, with Ki values generally in the micromolar range. The Ahx-Phe-Leu-Arg-COOH analogue was selected for conjugation to inorganic oxide nanoparticles and agarose gel beads. All conjugates exhibited inhibitory activity in the same range as for the free peptides. 

A serine protease inhibitor was extracted from potato tubers. The inhibitor was conjugated to soluble, prefractionated dextran and titanium dioxide and zinc oxide nanoparticles. Conjugation to dextran was achieved by periodate oxidation of the dextran, followed by Schiff base coupling to inhibitor amino groups, and finally reduction, whereas the conjugation to the oxide particles was carried out by aminosilanization and carbonyldiimidazole activation. The inhibitory effect of the conjugated inhibitor was compared to that of free inhibitor in solution and with gelatin gel as a direct substrate. A certain degree of inhibitory activity was retained for both the dextran-conjugated and particle-conjugated inhibitors. In particular, the apparent Ki value of the dextran-conjugated inhibitor was found to be in the same range as that for free inhibitor. The dextran conjugate retained a higher activity than the free inhibitor after 1 month of storage at room temperature.

The interaction between pancreatic proteases and a serine protease inhibitor purified from potato tubers was investigated by chromatography-coupled light scattering measurements. The molar mass distribution in the chromatogram was compared to theoretical values calculated for the different possible combinations of complexes and free components by three different approaches, namely section analyses of the chromatograms, full mass average determination and mass distribution analysis. This revealed that the inhibitor was able to bind trypsin in a 2:1 complex, whereas the data for chymotrypsin clearly showed a limitation to 1:1 complex regardless of the molar ratio in the injected samples. The same experiment carried out with elastase and the potato inhibitor gave only weak indications of complex formation under the conditions used.

Mass spectrometry-based proteomics benefits from efficient digestion of protein samples. In this study, trypsinwas immobilized on nanoporous anodized alumina membranes to create an enzyme reactor suitable for peptidemassfingerprinting. The membranes were derivatized with 3-aminopropyltriethoxysilane and the amino groupswere activated with carbonyldiimidazole to allow coupling of porcine trypsin viaε-amino groups. The functionwas assessed using the artificial substrate Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride, bovine ribonu-clease A and a human plasma sample. A 10-membraneflow-through reactor was used for fragmentation and MSanalysis after a single pass of substrate both by collection of product and subsequent off-line analysis, and bycoupling on-line to the instrument. The peptide pattern allowed correct identification of the single target proteinin both cases, and of > 70 plasma proteins in single pass mode followed by LC-MS analysis. The reactor retained76% of the initial activity after 14 days of storage and repeated use at room temperature.

Significance:This manuscript describes the design of a stable enzyme reactor that allows efficient and fast di-gestion with negligible leakage of enzyme and enzyme fragments. The high stability facilitates the use in anonline-setup with MS detection since it allows the processing of multiple samples within an extended period of time without replacement.

A model based on gelatin for protease activity studies was designed. The model is also extended to study the efficiency of inhibitors in a separate protective layer covering the layer containing the target substrate. A good correlation between protease concentration and the size of erosion wells formed in a plain gelatin layer was observed. Similarly, increased concentration of inhibitors gave a systematic decrease in well area. Kinetic analyses of the two-layer model in a spectrophotometric plate reader with a fixed concentration of substrate in the bottom layer displayed a strict dependence of both inhibitor concentration and thickness of the top "protective" layer. An apparent, but weaker inhibition effect was also observed without inhibitors due to diffusional and erosion delay of enzyme transport to the substrate-containing layer.

The previously uncharacterized Drosophila melanogaster Epsilon class glutathione transferases E6 and E7 were immobilized on nanoporous alumina. The nanoporous anodized alumina membranes were derivatized with 3-aminopropyl-triethoxysilane and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes via ∊-amino groups. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzyme in solution. A limited kinetic study was also carried out on immobilized human GST S1-1 (also known as hematopoietic prostaglandin D synthase). The stability of the immobilized enzymes was virtually identical to that for enzymes in solution and no leakage of enzyme from the matrix could be observed.

Nanoporous alumina membranes were silanized with aminopropylsilane and iminodiacetic acid (IDA) groups were generated in situ by reaction with iodoacetate. The membranes were mounted in standard filter holders, connected to a HPLC system and saturated with selected metal ions. Cu(II) allowed the capture of chicken muscle lactate dehydrogenase with such stability, repeatability and reproducibility that Michaelis-Menten kinetics could be studied. The IDA surface was stable for months and could be depleted and regenerated with metal ions multiple times without appreciable loss of capacity. The binding of lactate dehydrogenase influenced the backpressure to the extent that could be expected for a monolayer according to Poiseuilles law.