Response surface methodology for optimizing the bovine serum albumin fibrillation.
Amyloid fibrils are considered as nanostructures that could be formed by ordered self-assembly of the partially-folded states of many different peptides or proteins. In this study, bovine serum albumin was used as a model protein whose ordered aggregation (fibrillation) was optimized. Response surface methodology (RSM) was used in a design that contained a total of 30 experimental trials. The first 24 were organized in a factorial design and from 25 to 30 involved the replications of the central points. Data obtained from RSM were subjected to the analysis of variance (ANOVA) and analyzed using a second order polynomial equation. Subsequent testing of the suggested experimental parameters was done in vitro with Congo red spectrophotometric assay. Protein concentration, pH, temperature and time of incubation were the variables used in this study. Responses were assessed by measuring absorbance in 540 nm (characteristic of amyloid formation) and maximal wavelength. Concomitant effects of variables were assessed in surface plots that each considered two of the variables. Interestingly, the pattern obtained by monitoring absorbance at 540 nm and absorbance in maximal wavelength were identical in most cases. We are reporting the optimum concentration of protein, pH, temperature and time at 5 mg ml⁻¹, 3.02 and 72 °C and 48 h, respectively. Our findings suggest that use of Congo red spectrophotometric test, as a simple and affordable assay could be suggested as a first test for assessing fibril formation of proteins. Absorbance in maximal wavelength is recommended as a significant indication of fibril formation.
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. a.arasteh@srbiau.ac.ir
Amir
A
Arasteh
Mehran
M
Habibi-Rezaei
Azadeh
A
Ebrahim-Habibi
Ali Akbar
AA
Moosavi-Movahedi
D000682:Amyloid; D000704:Analysis of Variance; D000818:Animals; D002417:Cattle; D004396:Coloring Agents / Q000737:chemistry; D003196:Computer Graphics; D003224:Congo Red / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors
D000682:Amyloid; D004396:Coloring Agents; D012710:Serum Albumin, Bovine; D003224:Congo Red