Quantitation of hyaluronidases by the Morgan–Elson reaction: comparison of the enzyme activities in the plasma of tumor patients and healthy volunteers

Muckenschnabel, I.; Bernhardt, Günther; Spruß, Thilo; Dietl, B.; Buschauer, Armin

doi:10.1016/s0304-3835(98)00196-7

Cited by 61 publications

(49 citation statements)

References 35 publications

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“…Total bee venom, purified bee venom Hyase [23] as well as bovine testes Hyase (Sigma) were used as positive controls. Hyaluronic acid (Sigma) degradation was measured using the BSA-test and the Morgan-Elson assay as described previously [24][25][26]. The latter assay was also performed with the substrates chondroitin sulfate A and chondroitin sulfate C (Sigma).…”

Section: Purification and Activity Assays Of Recombinant Vespula Hyasesmentioning

confidence: 99%

A proteomic study of the major allergens from yellow jacket venoms

et al. 2007

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The venoms of stinging insects belong to the most dangerous allergen sources and can cause fatal anaphylactic reactions. Reliable prediction of a patient's risk to anaphylactic reactions is vital, and diagnosis requires the knowledge of the relevant allergens. Recently, a new hyaluronidase -like glycoprotein from Vespula vulgaris (Ves v 2b) was identified. This led us to investigate hyaluronidases and also other major allergens from V. germanica and four additional Vespula species. By MALDI-Q-TOF-MS, the new hyaluronidase-like protein was shown to be the major component of the 43-kDa band in all Vespula species studied. LC-ESI-Q-TOF-MS/MS sequencing of Ves g 2a and Ves g 2b facilitated the cloning of their cDNA. Ves v 2b and Ves g 2b turned out to be essentially identical on protein level. Whereas the less abundant "a" form displayed enzymatic activity, the new "b" homologue did not. This is probably caused by amino acid exchanges in the active site, and it raises questions about the physiological role of this protein. Sequence comparisons by MS/MS of antigen 5 and phospholipases from V. vulgaris, germanica, maculifrons, pensylvanica, flavopilosa and squamosa revealed the latter as a taxonomic outlier and led to the discovery of several not previously reported amino acid differences.

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Section: Purification and Activity Assays Of Recombinant Vespula Hyasesmentioning

confidence: 99%

A proteomic study of the major allergens from yellow jacket venoms

et al. 2007

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“…If these sugars converts from the pyranoside into the furanoside form and are boiled under alkaline conditions, a Morgan-Elson reaction can take place. One or more of the resulting chromogens reacts with DMAB under acidic conditions [40] and gives a red-colored product with a maximum in absorbance at 590 nm. Pyridines in general show good water solubility, because the free electron pair of the nitrogen is able to interact with polar water molecules at neutral pH.…”

Section: Chemistrymentioning

confidence: 99%

Indole carboxamides inhibit bovine testes hyaluronidase at pH 7.0 and indole acetamides activate the enzyme at pH 3.5 by different mechanisms

Kaessler

Nourrisson

Duflos

et al. 2008

Journal of Enzyme Inhibition and Medicinal Chemistry

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Hyaluronidases are enzymes controlling many crucial physiological processes. Imbalanced enzymatic activity is connected with severe diseases. Because there is limited availability of drugs modulating hyaluronidase activity, the search for hyaluronidase interacting compounds is getting more and more important. A series of fifteen indole carboxamides and acetamides were synthesized and tested on inhibition of bovine testes hyaluronidase. In vitro assays were performed using stains-all at pH 7 and the Morgan-Elson reaction at pH 3.5. At neutral pH, the most active inhibitory compound was N-(Pyridin-4yl)-[5-bromo-1-(4-fluorobenzyl)indole-3-yl]carboxamide (20) with an IC 50 value of 46 mM. Surprisingly, inhibition of all compounds was completely abolished by a decrease in pH. At pH 3.5 the activity of the enzyme was increased up to 134% by compound N-(4,6-Dimethylpyridin-2yl)-(1-ethylindole-3-yl)acetamide (24) at a concentration of 100 mM. The known activating effect of bovine serum albumine (BSA) on hyaluronidase activity was verified in the assay and compared to the effect of compound 24. Structure-activity relationships are discussed and a model is proposed, which explains the increase in activity at pH 3.5 by bonding of the protonated form of N-(4,6-Dimethylpyridin-2yl)-(1-ethylindole-3-yl)-acetamide (24) to hyaluronic acid. The bonding results in an elongated form of the substrate with easier enzymatic access.

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“…One of the most frequently used colorimetric assays is based on the measure of the concentration of reducing ends appearing during hydrolysis. In the Morgan-Elson assay (17), one of the first used, the N-acetyl-D-glucosamine reducing end is successively transformed into chromogens I and II under alkaline conditions at 100°C and then into chromogen III by the action of concentrated hydrochloric and sulfuric acids, and finally chromogen III reacts with p-dimethyaminobenzaldehyde (DMAB) (Ehrlich's reagent) to give a red colored product which can be detected at 585 nm (19). Reissig et al (18) have improved this assay by adding borate in the first step to increase the production of chromogens and thus to enhance the signal at 585 nm.…”

Section: Hyaluronan (Ha)mentioning

confidence: 99%

“…This procedure has been used, for example, to study HAase activity as a function of pH or ionic strength (10, 20 -24). However, a non-zero absorbance at the first time point following addition of HAase has been reported in some cases by those performing kinetic measurements (19).…”

Section: Hyaluronan (Ha)mentioning

confidence: 99%