Costa Tássia R, Carone Sante E I, Tucci Luiz F F, Menaldo Danilo L, Rosa-Garzon Nathalia G, Cabral Hamilton, Sampaio Suely V
1Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo (FCFRP-USP), Avenida do Café, s/n, B. Monte Alegre, Ribeirão Preto, SP CEP 14040-903 Brazil.
2Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo (FCFRP-USP), Ribeirão Preto, SP Brazil.
J Venom Anim Toxins Incl Trop Dis. 2018 Dec 4;24:37. doi: 10.1186/s40409-018-0172-9. eCollection 2018.
L-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 LAAOs, one isolated from (BjussuLAAO-II) and the other from (BmooLAAO-I) venoms.
The 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, Zn, Al, Cu and Ni 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.
Our 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.
从蛇毒中分离出的L-氨基酸氧化酶（SV-LAAOs）是具有巨大治疗潜力的酶，目前正作为开发治疗包括癌症和细菌感染在内的各种疾病新策略的工具进行研究。本研究的主要目的是对两种LAAOs的酶稳定性进行简要评估，一种从（BjussuLAAO-II）毒液中分离，另一种从（BmooLAAO-I）毒液中分离。
通过微孔板比色法评估两种LAAOs的酶活性和稳定性，为此将BjussuLAAO-II和BmooLAAO-I与不同的L-氨基酸底物在不同离子存在下、不同pH范围和温度下孵育。BjussuLAAO-II和BmooLAAO-I对疏水性氨基酸如苯丙氨酸和亮氨酸表现出更高的亲和力。这两种酶在25至75°C的宽温度范围内显示出高酶活性，并且在pH约为7.0时呈现最佳活性。此外，锌、铝、铜和镍离子对两种LAAOs的酶活性有负调节作用。关于稳定性，BjussuLAAO-II和BmooLAAO-I在4°C的中性pH溶液中储存42天时显示出高酶活性。此外，通过毛细管电泳分析了两种LAAOs的聚糖部分，结果显示BjussuLAAO-II呈现两个主要聚糖部分，相对质量分别为7.78和8.13 CGU，而BmooLAAO-I显示出7.58、7.94和8.37 CGU的三个部分。
我们的结果表明，在适当储存时，BjussuLAAO-II和BmooLAAO-I具有长时间的酶稳定性，这对于在医学领域具有重大影响的药理学研究中使用这些酶非常重要。
