Chromogenic substrate from 4-nitro-1-naphthol for hydrolytic enzyme of neutral or slightly acidic optimum pH: 4-Nitro-1-naphthyl-β-d-galactopyranoside as an example

2013 ◽  
Vol 23 (3) ◽  
pp. 646-649 ◽  
Author(s):  
Jizheng Dang ◽  
Hongbo Liu ◽  
Xiaolan Yang ◽  
Yi Zhang ◽  
Yanling Xie ◽  
...  
2010 ◽  
Vol 76 (19) ◽  
pp. 6485-6493 ◽  
Author(s):  
Benjamin L. Turner

ABSTRACT Extracellular enzymes synthesized by soil microbes play a central role in the biogeochemical cycling of nutrients in the environment. The pH optima of eight hydrolytic enzymes involved in the cycles of carbon, nitrogen, phosphorus, and sulfur, were assessed in a series of tropical forest soils of contrasting pH values from the Republic of Panama. Assays were conducted using 4-methylumbelliferone-linked fluorogenic substrates in modified universal buffer. Optimum pH values differed markedly among enzymes and soils. Enzymes were grouped into three classes based on their pH optima: (i) enzymes with acidic pH optima that were consistent among soils (cellobiohydrolase, β-xylanase, and arylsulfatase), (ii) enzymes with acidic pH optima that varied systematically with soil pH, with the most acidic pH optima in the most acidic soils (α-glucosidase, β-glucosidase, and N-acetyl-β-glucosaminidase), and (iii) enzymes with an optimum pH in either the acid range or the alkaline range depending on soil pH (phosphomonoesterase and phosphodiesterase). The optimum pH values of phosphomonoesterase were consistent among soils, being 4 to 5 for acid phosphomonoesterase and 10 to 11 for alkaline phosphomonoesterase. In contrast, the optimum pH for phosphodiesterase activity varied systematically with soil pH, with the most acidic pH optima (3.0) in the most acidic soils and the most alkaline pH optima (pH 10) in near-neutral soils. Arylsulfatase activity had a very acidic optimum pH in all soils (pH ≤3.0) irrespective of soil pH. The differences in pH optima may be linked to the origins of the enzymes and/or the degree of stabilization on solid surfaces. The results have important implications for the interpretation of hydrolytic enzyme assays using fluorogenic substrates.


1975 ◽  
Author(s):  
P. Friberger ◽  
G. Axelsson ◽  
K. Korsan-Bengtsen

Plasmin splits the chromogenic substrate B2-Phe-Val-Arg-pNA (S-2160, Bofors) at a relatively high rate. Standard plasmin in glycerol obtained from Nat. Inst, for Biol. Stand, and Contr., London, was tested in a system with Tris buffer of varying pH and ionic strength. The pH optimum for the reaction was found to be 7.4. Variations in ionic strength between 0.05–0.1 had insignificant influence but at higher ionic strength there was a slight inhibition. A linear relationship was found between plasmin and AOD/min. At optimum pH and a final substrate concentration of 0.2 mM 0.1 CTA unit corresponds to approximately 0.10 nkat. Purified plasminogen (AB Kabi, Stockholm, Sweden) in the concentrations 0.02–0.2 CU/ml was activated optimally with streptokinase (Kabikinase® ) in the concentrations 500–2000 IU/ml. Higher concentration gave inhibition. The activity of streptokinase activated plasminogen increased with a decreasing ionic strength. A linear relationship was found between streptokinase activated plasminogen and AOD/min. Approximately 3,000 Plong/units per ml of urokinase was needed to obtain the same activation as with optimal streptokinase concentration. The method has been used for determination of plasminogen in plasma. With final dilution of plasma in the range 1/20–1/200 activated by streptokinase (2000 IU/ml) in a system of pH 8.2, I = 0.05, a linear relationship was found between plasma dilution and AOD/min. The reproducibility in a series of tests is good (variation coefficient < 3%) and with insignificant interference by inhibitors. The determinations were easily carried out in a simple spectrometer (405 nm) and in an automatic reaction rate analyzer (LKB 8600, 410 nm).


2002 ◽  
Vol 68 (3) ◽  
pp. 1297-1304 ◽  
Author(s):  
C. Caldas ◽  
A. Cherqui ◽  
A. Pereira ◽  
N. Simões

ABSTRACT Xenorhabdus nematophila, a bacterium pathogenic for insects associated with the nematode Steinernema carpocapsae, releases high quantities of proteases, which may participate in the virulence against insects. Zymogram assays and cross-reactions of antibodies suggested that two distinct proteases were present. The major one, protease II, was purified and shown to have a molecular mass of 60 kDa and an estimated isoelectric point of 8.5. Protease II digested the chromogenic substrate N-tosyl-Gly-Pro-Arg-paranitroanilide (pNA) with V max and Km values of 0.0551 μM/min and 234 μM, respectively, and the substrate dl-Val-Leu-Arg-pNA with V max and Km values of 0.3830 μM/min and 429 μM, respectively. Protease II activity was inhibited 93% by Pefabloc SC and 45% by chymostatin. The optimum pH for protease II was 7, and the optimum temperature was 23°C. Proteolytic activity was reduced by 90% at 60°C for 10 min. Sequence analysis was performed on four internal peptides that resulted from the digestion of protease II. Fragments 29 and 45 are 75 and 68% identical to alkaline metalloproteinase produced by Pseudomonas aeruginosa. Fragment 29 is 79% identical to a metalloprotease of Erwinia amylovora and 75% identical to the protease C precursor of Erwinia chrysanthemi. Protease II showed no toxicity to hemocytes but destroyed antibacterial activity on the hemolymph of inoculated insects' larvae and reduced 97% of the cecropin A bacteriolytic activity.


1989 ◽  
Vol 54 (8) ◽  
pp. 2276-2286
Author(s):  
Tsezengijn Dash ◽  
Tomislav Barth ◽  
Jiřina Slaninová ◽  
Jana Barthová ◽  
Hana P. Mašková ◽  
...  

A reproducible method has been developed for the isolation of the adenohypophyseal enzyme with a trypsin-like activity. The enzyme is able to hydrolyze Nα-benzoyl-L-arginine-p-nitroanilide, a fluorogenic substrate CBzl-Arg-Arg-β-naphthyl amide and some peptides with one or two accumulated basic amino acids in the chain. The optimum pH for hydrolysis of the chromogenic substrate was within the range 6.0-7.0 (Km = 0.66 mmol l-1), in the case of the fluorogenic substrate the range was between 7.0 and 7.5 (Km = 1.2 μmol l-1). The enzyme is activated by cysteine and dithiothreitol and inhibited by SH-poisons. The molecular weight of the enzyme, determined by means of two independent methods, was approximately 25 kDA.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maiko Uehara ◽  
Eri Tabata ◽  
Mikoto Okuda ◽  
Yukari Maruyama ◽  
Vaclav Matoska ◽  
...  

AbstractDiet of the crab-eating monkey (Macaca fascicularis) consists of both plants and animals, including chitin-containing organisms such as crabs and insects. This omnivorous monkey has a high expression of acidic chitinase (CHIA) in the stomach and here, we report on its enzymatic properties under different conditions. When we compared with Mus musculus CHIA (Mm-CHIA), Macaca fascicularis CHIA (Mf-CHIA) exhibits higher chitinolytic activity at broad pH (1.0–7.0) and temperature (30–70 ℃) range. Interestingly, at its optimum pH (5.0), Mf-CHIA showed the highest activity at 65 °C while maintaining it at robust levels between 50 and 70 °C. The degradation efficiency of Mf-CHIA was superior to Mm-CHIA toward both polymeric chitin as well as an artificial chromogenic substrate. Our results show that unique features of Mf-CHIA including its thermostability warrant the nomination of this enzyme for potential agricultural and biomedical applications.


2013 ◽  
Vol 12 (2) ◽  
pp. 11 ◽  
Author(s):  
Abdul Wahab Mohammad ◽  
Jamaliah Md.Jahim ◽  
Suhaila Johar ◽  
Osman Hassan

Cutinase is a hydrolytic enzyme that has both properties of lipase and esterase, thus finding its use in many areas. Previous studies have investigated both upstream and downstream processes for cutinase production from microbial source. However, no study has yet to address the use of membrane chromatography for cutinase purification, which is more favourable in terms of process resolution and product throughput as compared to the conventional gel chromatography. Hydrophobic interaction was chosen as the separation mechanism for cutinase purification in this study. The optimisation of cutinase purification in two different types of chromatographic media; conventional packed-gel and membrane matrix, were pre-determined by the best compromise between the recovery and purity of the purified cutinase. It was found that the optimised condition were of pH 4.0 and 1.0 M ammonium sulfate for the conventional column (50% recovery, 4.8-fold purity) and pH 6.0 with 1.5 M ammonium sulfate for the membrane–matrix column(87% recovery, 30-fold purity). Preferential interaction analysis was used to describe the protein chromatographic behaviour in each chromatographic media. Graph of natural algorithm of protein retention data to the function of salt concentration at pH 4.0 and 6.0 for each column were plotted. It was found that at the optimum pH condition for gel-packed column, a small amount of ammonium sulfate was sufficient to achieve maximum cutinase recovery and purity since the effect of salt at that particular pH were less significant. Consequently, the number of released water molecules were calculated and it was observed that for membrane column, larger number of water was released at pH 6.0 illustrating more protein were bounded to the stationary phase, thus explaining the optimum pH condition of the particular column.


1998 ◽  
Vol 79 (01) ◽  
pp. 87-90 ◽  
Author(s):  
D. W. Jones ◽  
M. Winter ◽  
M. J. Gallimore

SummaryFactor XII (FXII) levels were determined in plasma samples from 29 normal donors, 10 patients with inherited FXII deficiency (all lupus anticoagulant [LA] negative) and 67 LA positive patients, using clotting (FXIIct), chromogenic substrate (FXIIcs) and immunochemical (FXIIag) assays. Excellent correlations were obtained in the three FXII assays with the LA negative samples and between the FXIIcs and FXIIag assays in the LA positive samples. Correlations between both the FXIIcs and FXIIag with FXIIct in the LA positive patients were poor. Of 67 LA positive samples studied, 25 (37.3%) showed lower values in the FXIIct assay; 13 (19.4%) of these patients were pseudo FXII deficient with values of FXII below the lower limit of normal.These results indicate that a diagnosis of FXII deficiency can be made inappropriately in the presence of phospholipid antibodies and that such a diagnosis should not be made by FXIIct assay alone.


1986 ◽  
Vol 55 (02) ◽  
pp. 276-278 ◽  
Author(s):  
F Brosstad ◽  
Inge Kjønniksen ◽  
B Rønning ◽  
H Stormorken

SummaryA method for visualization of the multimeric forms of von Willebrand Factor (vWF) in plasma and platelets is described. The method is based upon: 1) Separation of the vWF multimers by SDS-agarose electrophoresis, 2) Subsequent blotting of the vWF multimers onto nitrocellulose, 3) Immunolocalization and visualization of the vWF pattern by the sequential incubation of the blot with a) primary vWF antiserum, b) peroxidase- or beta-galactosidase-conjugated secondary antibodies and a relevant chromogenic substrate.


1979 ◽  
Vol 41 (03) ◽  
pp. 544-552 ◽  
Author(s):  
R P Herrmann ◽  
P E Bailey

SummaryUsing the chromogenic substrate, Tos-Gly-Pro-Arg-pNA-HCL (Chromozym TH, Boehringer Mannheim) plasma thrombin was estimated in six cases of envenomation by Australian elapid snakes. All patients manifested findings chracteristic of defibrination due to envenomation by these snakes. Fibrin-fibrinogen degradation products were grossly elevated, as was plasma thrombin in all cases.Following treatment with antivenene, all abnormal coagulation parameters returned rapidly towards normal by 24 hours and plasma thrombin disappeared.


1989 ◽  
Vol 61 (03) ◽  
pp. 386-391 ◽  
Author(s):  
Guido Tans ◽  
Truus Janssen-Claessen ◽  
Jan Rosing

SummaryIn this paper we report a method via which enzymatically active products formed during prothrombin activation can be detected by simple photographic means after SDS-gel electrophoresis, blotting onto a nitrocellulose membrane and visualization with the chromogenic substrate, S2238. After amidolytic detection the same nitrocellulose membrane can also be used for immunologic detection of prothrombin activation products, thus allowing a complete description of product formation during prothrombin activation.The detection limit of the so-called “amidoblot” is approximately 3 ng thrombin per gel sample which is comparable to the sensitivity of immunoblotting.It is further shown that the amidoblot technique can also be applied to other coagulation factors for which a suitable chromogenic substrate is available (factor XIIa, kallikrein, factor XIa, factor Xa, plasmin and activated protein C).


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