Validation of Modified Lowry for the Determination of Total Protein Concentration of Rhopalurus junceus Scorpion Venom

the Determination of Total Protein Concentration of Rhopalurus junceus Scorpion is mainly composed by proteins and peptides. The determination of total protein has been determined by Lowry method. However, the reading has been performed in a spectrophotometer using quartz cuvettes. In the present study, we validate the modified Lowry method for the determination of total protein concentration of Rhopalurus junceus scorpion venom in microplates. The validated parameters were linearity, range, accuracy, repeatability, and intermediate precision. This method was specific for the quantification of total protein concentration of Rhopalurus junceus scorpion venom. The curve of modified Lowry was lineal in the evaluated range of 0.02- 0.18 mg/mL (R 2 = 0.998). The method was accurate with recovery percentage of 100 ± 10% and possessed a good intra-day and inter-day precision based on relative standard deviation values (less than 4.3% and 9%, respectively). The validation was satisfactory and the method of modified Lowry to microplates is considered valid for the quantification of total proteins in Rhopalurus junceus scorpion venom.


Validation of Modified Lowry for the Determination of Total Protein Concentration of Rhopalurus junceus
Scorpion Venom Introduction Scorpion venoms are a complex mixture of peptides and proteins with a broad range of biological activities, among which are peptides that impair proper functioning of ion-channels [1].
The molecules and peptides from the scorpion venom can be used to develop highly potent anticancer, antimicrobial and many other effective drugs [2]. Over the past few years, increasing number in vitro and in vivo studies with scorpion venom have been shown it capacity of the inhibition of cancer progression and induce apoptosis of tumor cells [3].
Rhopalurus junceus (R. junceus) scorpion is an endemic species of Cuba that belongs to the Buthidae family and the venom from this scorpion has been used in traditional medicine as an anti-inflammatory, analgesic and antitumor drug [4]. Previous preclinical studies from our group have shown that R.junceus scorpion venom exerts a selective and cytotoxic effect against a panel of epithelial cancer cells without affecting normal cells [5].
Besides, this scorpion venom has been able to induce apoptosis on HeLa cervical-uterine cancer [5] and MDA-MB-231 metastatic breast cancer cell lines [6].
The validation is an important procedure in the pharmaceutical industry and is used to ensure that quality is built into the processes supporting drug development and manufacture [7]. Although many assays exist to determine total protein content in a variety of sample matrices, the first approach is the Lowry assay, which is based on formation of protein-copper complexes and subsequent reduction with Folin-Ciocalteu Reagent [8]. In our entrepreneurial group, the concentration of total proteins is determined by the Lowry method.
In this conventional technique for measuring protein concentration,

Analytical Method
The concentration of proteins present in R. junceus scorpion venom was determined through the method of modified Lowry [9].
Bovine Serum Albumin (BSA) was used as the reference standard.

Validation Parameters
The validation parameters selected are in correspondence with those defined in international guides such as the International

Specificity
For the specificity study we analyzed distilled water (placebo) and samples of Rhopalurus junceus scorpion venom. Absorbance values at 620 nm were determined and the absence of interferences verified in the analytic answer.

Linearity and Interval of the System
Six experiments were carried out in total, three independent experiments by two analysts and the corresponding calibration curves were constructed. The concentrations used of BSA were:

Accuracy
It was performed by evaluating the addition of BSA standard to the sample of R. junceus scorpion venom [10]. Samples of R.
junceus scorpion venom was diluted 1:100, which it was prepared at a concentration such that its absorbance corresponded to the

Results and Discussion
The specificity of the method was demonstrated by the analysis of the blank matrix (H 2 O), which did not absorb at the working wavelength, so the method was specific for the quality control of Each sample was analyzed three times. Good linearity was obtained ( Figure 1). The relationship between the calibration curve and regression coefficient was: A=0.9896 C+ 0.0015 and correlation coefficient (R 2 ) was higher than 0.98, indicating good linearity in the proposed range [10]. The statistical results of the calibration curves are presented in Table 1.  The five points of the calibration curve showed accuracy within the specified parameters. For intra-test and inter-test accuracy, the acceptance criteria established were RSD ≤ 5% and RSD ≤ 10%, respectively ( Table 2). For accuracy, recovery percentages between 100 ± 10% were obtained. All points of the curve accomplished the acceptance criteria established for the Student t test, since the tcalc ≤ tteo for a 95% confidence [10]. The Table 3 shows the mean recoveries for three ranges (LR, MR and HR). The results were was 100 ± 3%, 106 ± 5% and 94 ± 3% and the RSD was less than 5%. In addition, all points of the curve met the acceptance criteria established for the Student's t-test, since the tcalc ≤ tteo for a 95% confidence. These results demonstrated the accomplished the acceptance criteria established for linearity, but now with 3 points       The results in the intermediate precision studies are shown in Table 5. The concentration values obtained by two analysts on three different days, showed RSD below of 10% established as a maximum limit, which demonstrates compliance with the acceptance criteria in the entire range studied for R. junceus scorpion venom [10]. The Lowry technique seems to be the best method in determining the protein concentration of hen egg, because Biuret assay is not much sensitive and Bradford can be inhibited by the presence of many compounds [11]. For that reason, our entrepreneurial group validated the Lowry technique for determination of total protein in R.junceus scorpion venom and proposes the implementation of the Lowry method modified to microplate in our quality department.
Besides, the present study could be very useful for another group that work with scorpion venom or for other studies that need to validate one analytical method for changing of equipment to measure the concentration of one analyte [12][13][14].

Conclusion
The method of Lowry modified to microplates is specific, linear, precise and exact, so it is considered valid for the quantification of proteins present in R. junceus scorpion venom.