Kinetic investigations and stability studies of two Bothrops L-amino acid oxidases

Abstract

BackgroundL-amino acid oxidases isolated from snake venoms (SV-LAAOs) are enzymes that have great therapeutic potential and are currently being investigated as tools for developing new strategies to treat various diseases, including cancer and bacterial infections. The main objective of this study was to make a brief evaluation of the enzymatic stability of two Bothrops LAAOs, one isolated from Bothrops jararacussu (BjussuLAAO-II) and the other from Bothrops moojeni (BmooLAAO-I) venoms.Methods and resultsThe enzymatic activity and stability of both LAAOs were evaluated by microplate colorimetric assays, for which BjussuLAAO-II and BmooLAAO-I were incubated with different L-amino acid substrates, in the presence of different ions, and at different pH ranges and temperatures. BjussuLAAO-II and BmooLAAO-I demonstrated higher affinity for hydrophobic amino acids, such as Phe and Leu. The two enzymes showed high enzymatic activity in a wide temperature range, from 25 to 75 °C, and presented optimum pH around 7.0. Additionally, Zn2+, Al3+, Cu2+ and Ni2+ ions negatively modulated the enzymatic activity of both LAAOs. As to stability, BjussuLAAO-II and BmooLAAO-I showed high enzymatic activity for 42 days stored at 4 °C in neutral pH solution. Moreover, the glycan portions of both LAAOs were analyzed by capillary electrophoresis, which revealed that BjussuLAAO-II presented two main glycan portions with relative masses of 7.78 and 8.13 CGU, while BmooLAAO-I showed three portions of 7.58, 7.94 and 8.37 CGU.ConclusionsOur results showed that, when stored properly, BjussuLAAO-II and BmooLAAO-I present enzymatic stability over a long time period, which is very important to allow the use of these enzymes in pharmacological studies of great impact in the medical field.

Bothrops moojeni L-amino acid oxidase induces apoptosis and epigenetic modulation on Bcr-Abl+ cells

“…Natural compounds, including SV-LAAO, have exhibited strong antitumor activity [ 22 , 28 - 30 , 32 , 40 ]. In the present study, BmooLAAO-I decreased cell viability of all the leukemic cell lines tested: HL-60, HL-60.Bcr-Abl, K562-S, and K562-R. To address whether H 2 O 2 released during the LAAO enzymatic activity of BmooLAAO-I mediated cell death, we carried out the cytotoxicity assay in the presence of 100 µg/mL catalase.…”

“…The LAAO isolated from Bothrops moojeni (BmooLAAO-I) exerts antitumor action on Ehrlich ascites carcinoma cells and HL-60 acute promyelocytic leukemia cells [ 31 ]. The long-term enzymatic stability of BmooLAAO-I makes it possible to assess its pharmacological effects [ 32 ]. The present study examined whether BmooLAAO-I affected the apoptotic and epigenetic machineries of Bcr-Abl + cell lines resistant and responsive to imatinib mesylate.…”

Characterization of L-amino Acid Oxidase Derived from Crotalus adamanteus Venom: Procoagulant and Anticoagulant Activities

“…Furthermore, it was the direct deaminating action of LAAO, not the coincident generation of H 2 O 2 , that was responsible for the coagulation procoagulant profile as demonstrated by the GSH addition experiments. Further, the experiments involving CORM-2 demonstrated functionally, that the procoagulant effects of LAAO were not affected as was the phospholipase A 2 and raw venom activity of Crotalus adamanteus [6,7,8]. The lack of inhibition of the LAAO activity by EDTA and the transformation of procoagulant to anticoagulant activity by PMSF further confirmed that the phenomena observed in this system were LAAO mediated.…”

“…The previous works used solutions of isolated L-amino acids (L-Alanine, L-Aspartate, L-Glycine, L-Valine, etc.) to assess changes in specific LAAO activities [3,6,10], whereas this investigation assessed functional changes in coagulation. The EDTA experiments of the present work demonstrated no change in procoagulant activity by the LAAO, similar to the small amount of a decrease in activity in other LAAO [3,4,5].…”

“…Specifically, the range of inhibition of various LAAO activities by ethylenediaminetetraacetic acid (EDTA) was 0–30% whereas there was much greater variation observed with phenylmethylsulfonyl fluoride (PMSF), with inhibition observed at 20–90% [3,4,5]. The purified preparations of the LAAO are very stable in solution at 4 °C, without a significant change in activity for weeks [6]. In sum, snake venom LAAO are medically important enzymes that have unusual characteristics that are distinctly different from metalloproteinases and serine proteases.…”