Exploring purification methods to improve retrieval of collagenous binder residues from archeological murals

Abstract

A comparison was conducted on five methods for purifying collagenous residues extracted from paint replicas. The bicinchoninic acid method was used to quantify proteins recovered by these purification methods, and the conductivity in purified solutions was monitored. Statistical analysis was carried out by multivariate analysis of variance (MANOVA). The effect of different purification processes upon the molecular structure of the retrieved proteins was characterized with the aid of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), ultraviolet (UV) absorption spectroscopy, and circular dichroism (CD) spectroscopy. The results showed that the five methods all had significant desalination effects, among which dialysis, ultrafiltration, and ethanol precipitation could achieve lower conductivity. The protein recovery rate of dialysis was remarkably higher than the rates of other methods. With the aging time of the paint replicas, the recovery rates of all purification methods showed a downward trend, with the advantage of dialysis becoming more and more remarkable. No significant differences were found in the molecular weight and microstructure of the residues retrieved by the five purification methods, indicating that the impacts of different purification processes on the microstructure of proteins / peptides are approximately the same. A total of 29 identified peptides (consisting of 18 unique ones) corresponding to rabbit collagen were successfully detected in a model sample using the improved pretreatment. This paper concluded that dialysis is a more promising purification method for archeological paint samples with a higher potential to retrieve more residues for subsequent analysis.