Quantitative analysis and degradation mechanisms of different protein degradation methods

Abstract

The abrasive debris produced by wear test of artificial joints in vitro is encapsulated by proteins in serum lubricants, which hinder the characterization of debris analysis.One of the key issues of isolating wear debris from serum is degrading the proteins wrapping the wear debris. In this article, the proteins in calf serum were degraded by a strong alkali, a strong acid, and an enzyme. The residual concentration of proteins in calf serum was detected by UV absorption. Quantitative analysis of protein degradation and the protein degradation rate was proposed, following treatment with different degradation reagents and different incubation times. The results showed that when 10 mL of 25% volume calf serum was added with 40 mL of NaOH and incubated at 65 C for 24 h, the protein degradation rate reached a maximum of 95.52%.The protein degradation rate in the solution ranged from 31.86% to 71.64% when a different volume of 37% HCl was added and incubated at 60 C. The highest protein degradation rate was 94.98% in the protease degradation solution. When the protein degradation rate is less than 70%, the particles were coated by protein. When the protein degradation rate was more than 95%, there was no protein coating around the particles. The three protein degradation methods have different processes and protein degradation rates. A suitable method for protein degradation can be selected according to these practical applications.artificial joints, degradation rate, protein degradation, recovery of debris | INTRODUCTIONAfter an artificial joint is implanted into the human body, it is not only soaked by body fluids but is also impacted by and subjected to normal and shear forces. The abrasive particles generated by the wear of joints induce osteoclasts to produce osteolytic factors and induce bone resorption. Aseptic loosening of the prosthesis due to bone dissolution is the most common cause of the failure of the prosthesis. [1][2][3] The different sizes and morphologies of debris reflect the different wear behavior and mechanism of the artificial joint in addition to the different degrees of biological reaction. [4][5][6][7][8] Therefore, a reliable method of isolating debris from serum during artificial joint wear tests in vitro is important to the analysis of debris characteristics.Calf serum has been widely used as the lubricating fluid in vitro for wear tests of artificial joints. [9][10][11] Artificial joint debris binds to proteins in serum by ionization, hydrophobicity, and hydrophilicity. The joint debris is wrapped by multiple layers of proteins called the protein halo. 12-14 Therefore, the degree of protein degradation affects the separation and analysis of the amount and morphology of the debris.A suitable protein degradation method is an important prerequisite for the isolation and analysis of wear debris.Many scholars have researched different protein degradation methods. Different protein degradation methods have different requirements for the amount of strong acid, strong alkali and protease, temperat...

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“…The likely explanation could be attributed to the strong acidic properties of hydrochloric acid. The greater the deviation of the pH value from the pI values, the stronger the repulsion force within the protein tertiary structure, resulting in a more pronounced increase in the protein degradation rate [ 21 ]. Nevertheless, sodium hydroxide, despite being a strong alkali, still yielded a high content of crude proteins.…”

“…Nevertheless, sodium hydroxide, despite being a strong alkali, still yielded a high content of crude proteins. One potential explanation is that the non-ionized heme’s interaction with the distal histidine in the strong alkaline solution could reduce its ability to bind by oxidation, thereby partially inhibiting the oxidation process of the strong alkali, resulting in a longer duration of protein degradation in the strong alkali solution compared to an acidic one [ 21 ]. Furthermore, a remarkable enhancement in protein solubility was noted under alkaline conditions, particularly at pH 9 and above [ 22 ].…”