Francisco Cadena-Cadena, Luis Cárdenas-López José, Marina Ezquerra-Brauer Josafat, Javier Cinco-Moroyoqui Francisco, Alexis López-Zavala Alonso, Del Carmen Santacruz-Ortega Hisila, Alfredo Rivero-Espejel Ignacio
Departamento de Investigacion y Posgrado de Alimentos, Universidad de Sonora, Hermosillo, Sonora, Mexico.
Departamento de Ciencias Quimico Biologicas, Universidad de Sonora, Hermosillo, Sonora, Mexico.
Protein Pept Lett. 2019;26(7):532-541. doi: 10.2174/0929866526666190405124353.
Cathepsin D is a lysosomal enzyme that is found in all organisms acting in protein turnover, in humans it is present in some types of carcinomas, and it has a high activity in Parkinson's disease and a low activity in Alzheimer disease. In marine organisms, most of the research has been limited to corroborate the presence of this enzyme. It is known that cathepsin D of some marine organisms has a low thermostability and that it has the ability to have activity at very acidic pH. Cathepsin D of the Jumbo squid (Dosidicus gigas) hepatopancreas was purified and partially characterized. The secondary structure of these enzymes is highly conserved so the role of temperature and pH in the secondary structure and in protein denaturation is of great importance in the study of enzymes. The secondary structure of cathepsin D from jumbo squid hepatopancreas was determined by means of circular dichroism spectroscopy.
In this article, our purpose was to determine the secondary structure of the enzyme and how it is affected by subjecting it to different temperature and pH conditions.
Circular dichroism technique was used to measure the modifications of the secondary structure of cathepsin D when subjected to different treatments. The methodology consisted in dissecting the hepatopancreas of squid and freeze drying it. Then a crude extract was prepared by mixing 1: 1 hepatopancreas with assay buffer, the purification was in two steps; the first step consisted of using an ultrafiltration membrane with a molecular cut of 50 kDa, and the second step, a pepstatin agarose resin was used to purification the enzyme. Once the enzyme was purified, the purity was corroborated with SDS PAGE electrophoresis, isoelectric point and zymogram. Circular dichroism is carried out by placing the sample with a concentration of 0.125 mg / mL in a 3 mL quartz cell. The results were obtained in mdeg (millidegrees) and transformed to mean ellipticity per residue, using 111 g/mol molecular weight/residue as average. Secondary-structure estimation from the far-UV CD spectra was calculated using K2D Dichroweb software.
It was found that α helix decreases at temperatures above 50 °C and above pH 4. Heating the enzyme above 70°C maintains a low percentage of α helix and increases β sheet. Far-UV CD measurements of cathepsin D showed irreversible thermal denaturation. The process was strongly dependent on the heating rate, accompanied by a process of oligomerization of the protein that appears when the sample is heated, and maintained a certain time at this temperature. An amount typically between 3 and 4% α helix of their secondary structure remains unchanged. It is consistent with an unfolding process kinetically controlled due to the presence of an irreversible reaction. The secondary structure depends on pH, and a pH above 4 causes α helix structures to be modified.
In conclusion, cathepsin D from jumbo squid hepatopancreas showed retaining up to 4% α helix at 80°C. The thermal denaturation of cathepsin D at pH 3.5 is under kinetic control and follows an irreversible model.
组织蛋白酶D是一种溶酶体酶，存在于所有参与蛋白质周转的生物体中，在人类中，它存在于某些类型的癌组织中，在帕金森病中活性较高，而在阿尔茨海默病中活性较低。在海洋生物中，大多数研究仅限于证实这种酶的存在。已知一些海洋生物的组织蛋白酶D热稳定性较低，并且能够在非常酸性的pH值下保持活性。对巨型鱿鱼（茎柔鱼）肝胰腺中的组织蛋白酶D进行了纯化和部分特性分析。这些酶的二级结构高度保守，因此温度和pH值在二级结构和蛋白质变性中的作用在酶的研究中非常重要。通过圆二色光谱法测定了巨型鱿鱼肝胰腺中组织蛋白酶D的二级结构。
在本文中，我们的目的是确定该酶的二级结构以及将其置于不同温度和pH条件下时它是如何受到影响的。
使用圆二色技术测量组织蛋白酶D在接受不同处理时二级结构的变化。方法包括解剖鱿鱼的肝胰腺并进行冷冻干燥。然后通过将肝胰腺与测定缓冲液按1:1混合制备粗提物，纯化分两步进行；第一步使用截留分子量为50 kDa的超滤膜，第二步使用胃蛋白酶抑制剂琼脂糖树脂纯化该酶。酶纯化后，通过SDS-PAGE电泳、等电点和酶谱证实其纯度。将浓度为0.125 mg/mL的样品置于3 mL石英池中进行圆二色测量。结果以毫度（mdeg）获得，并转换为每个残基的平均椭圆率，使用111 g/mol分子量/残基作为平均值。使用K2D Dichroweb软件从远紫外CD光谱计算二级结构估计值。
发现在温度高于50°C和pH值高于4时α螺旋减少。将酶加热至70°C以上会使α螺旋的比例保持在较低水平并增加β折叠。组织蛋白酶D的远紫外CD测量显示出不可逆的热变性。该过程强烈依赖于加热速率，伴随着蛋白质在加热样品时出现的寡聚化过程，并在此温度下保持一定时间。其二级结构中通常有3%至4%的α螺旋保持不变。这与由于存在不可逆反应而受到动力学控制的解折叠过程一致。二级结构取决于pH值，pH值高于4会导致α螺旋结构发生改变。
总之，巨型鱿鱼肝胰腺中的组织蛋白酶D在80°C时仍保留高达4%的α螺旋。组织蛋白酶D在pH 3.5时的热变性受动力学控制并遵循不可逆模型。
