Determination of phospholipase A2 activity by a colorimetric assay using a pH indicator

Araújo, Albetiza Lôbo de; Radvanyi, François

doi:10.1016/0041-0101(87)90136-x

Cited by 160 publications

(48 citation statements)

References 13 publications

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“…Enzymatic activity of BaspPLA 2 -II was determined by the colorimetric method of de Araujo and Radvanyi [31], with phenol red as a pH indicator, upon micelles of 0.4% v/v Triton X-100 and 0.25% w/v sn-3-phosphatidylcholine as substrate. Twenty microliters of a solution of enzyme, containing 500, 250, 125, or 62.5 ng, in water, were added to 1 ml of substrate in a thermoregulated cuvette at 30 C. After a stabilization period of 20 s, the decrease in absorbance at 558 nm was monitored continuously for 1 min.…”

Section: Phospholipase a 2 Activitymentioning

confidence: 99%

Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops asper of Costa Rica: Biochemical and toxicological characterization☆

et al. 2010

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a b s t r a c tPhospholipases A 2 (PLA 2 ) are major components of snake venoms, exerting a variety of relevant toxic actions such as neurotoxicity and myotoxicity, among others. Since the majority of toxic PLA 2 s are basic proteins, acidic isoforms and their possible roles in venoms are less understood. In this study, an acidic enzyme (BaspPLA 2 -II) was isolated from the venom of Bothrops asper (Pacific region of Costa Rica) and characterized. BaspPLA 2 -II is monomeric, with a mass of 14,212 AE 6 Da and a pI of 4.9. Its complete sequence of 124 amino acids was deduced through cDNA and protein sequencing, showing that it belongs to the Asp49 group of catalytically active enzymes. In vivo and in vitro assays demonstrated that BaspPLA 2 -II, in contrast to the basic Asp49 counterparts present in the same venom, lacks myotoxic, cytotoxic, and anticoagulant activities. BaspPLA 2 -II also differed from other acidic PLA 2 s described in Bothrops spp. venoms, as it did not show hypotensive and anti-platelet aggregation activities. Furthermore, this enzyme was not lethal to mice at intravenous doses up to 100 mg (5.9 mg/g), indicating its lack of neurotoxic activity. The only toxic effect recorded in vivo was a moderate induction of local edema. Therefore, the toxicological characteristics of BaspPLA 2 -II suggest that it does not play a key role in the pathophysiology of envenomings by B. asper, and that its purpose might be restricted to digestive functions. Immunochemical analyses using antibodies raised against BaspPLA 2 -II revealed that acidic and basic PLA 2 s form two different antigenic groups in B. asper venom.

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Section: Phospholipase a 2 Activitymentioning

confidence: 99%

Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops asper of Costa Rica: Biochemical and toxicological characterization☆

et al. 2010

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“…They were previously reported in C. dioscoridis 20 , E. hirta 21 Acute toxicity (median lethal dose; LD 50 …”

Section: Resultsmentioning

confidence: 99%

“…The obtained results indicated that different doses of the investigated alcohol extracts (500, 1000, 2000, and 5000 mg/kg) did not produce any symptoms of acute toxicity and none of the mice died during 24 h of observation. It was suggested that oral LD 50 of was higher than 5000 mg/kg. Since substances possessing LD 50 higher than 50 mg/kg are non-toxic 26 , the tested extracts were considered safe.…”

Section: ) Testmentioning

confidence: 99%

“…It was suggested that oral LD 50 of was higher than 5000 mg/kg. Since substances possessing LD 50 higher than 50 mg/kg are non-toxic 26 , the tested extracts were considered safe.…”

Section: ) Testmentioning

confidence: 99%

“…Our main objective was to find an extract which was able to inhibit selectively the proinflammatory phospholipase A2 group IIA with no or minimal inhibitory effect on the digestive phospholipase A2 group IB. The test of inhibitory activity of PLA 2 was performed as described by De Aranjo and Radvany 50 . Briefly, the substrate consisted of 3.5 mM lecithin in a mixture of 3 mM NaTDC, 100 mM NaCl, 10 mM CaCl 2 , and 0.055 mM red phenol as a colorimetric indicator in 100 ml H 2 O.…”

Section: Inhibition Of Spla 2 Activitymentioning

confidence: 99%

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Prophylactic and curative anti-ulcerative colitis activity and the possible mechanisms of action of some desert plants

El‐Meligy

Awaad

Soliman

et al. 2014

Journal of Enzyme Inhibition and Medicinal Chemistry

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The aim of the present study was to evaluate both prophylactic and curative anti-ulcerative colitis activity and the possible mechanism of action of seven desert plant extracts. (Asclepiadaceae) were separately evaluated at three doses (125, 250, and 500 mg/kg) using the acetic acid-induced colitis model. The investigated extracts possessed prophylactic and curative anti-ulcerative colitis activities in a dose-dependent manner, where Salvia lanigera (87.9) and Solenostemma arghel (89.2) were the most effective extracts whereas the dexamesathone produced 68%. These extracts were further investigated for estimation of their mechanism of action. The in vitro potential radical (DPPH) scavenging activities of the investigated extracts were well supported with the reduction of colonic MDA content for both extracts. Suppression of the inflammatory mediator TNF-a and inhibition of both PLA2 and protease enzymes may play an important role in the anti-ulcerative colitis activities. The investigated extracts were safe for use up to 5 g/kg and the total alcohol extracts of Salvia lanigera and Solenostemma arghel (400 mg/kg for 35 d) showed no alteration on liver and kidney functions. Phytochemical screening of the investigated extracts revealed the presence of flavonoids, tannins, unsaturated sterols, and proteins which could be responsible for the activities.

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Proteomic characterization of the multiple forms of the PLAs from the venom of the social wasp Polybia paulista

et al. 2011

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The phospholipases A(1) (PLA(1) s) from the venom of the social wasp Polybia paulista occur as a mixture of different molecular forms. To characterize the molecular origin of these structural differences, an experimental strategy was planned combining the isolation of the pool of PLAs from the wasp venom with proteomic approaches by using 2-D, MALDI-TOF-TOF MS and classical protocols of protein chemistry, which included N- and C-terminal sequencing. The existence of an intact form of PLA(1) and seven truncated forms was identified, apparently originating from controlled proteolysis of the intact protein; in addition to this, four of these truncated forms also presented carbohydrates attached to their molecules. Some of these forms are immunoreactive to specific-IgE, while others are not. These observations permit to raise the hypothesis that naturally occurring proteolysis of PLA(1) , combined with protein glycosylation may create a series of different molecular forms of these proteins, with different levels of allergenicity. Two forms of PLA(2) s, apparently related to each other, were also identified; however, it was not possible to determine the molecular origin of the differences between both forms, except that one of them was glycosylated. None of these forms were immunoreactive to human specific IgE.

show abstract

Determination of phospholipase A2 activity by a colorimetric assay using a pH indicator

Cited by 160 publications

References 13 publications

Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops asper of Costa Rica: Biochemical and toxicological characterization☆

Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops asper of Costa Rica: Biochemical and toxicological characterization☆

Prophylactic and curative anti-ulcerative colitis activity and the possible mechanisms of action of some desert plants

Proteomic characterization of the multiple forms of the PLAs from the venom of the social wasp Polybia paulista

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