Analysis of variance was performed for all the data. The results indicated that buffer composition (p < 0.001), storage temperature (p = 0.04), and addition of BIOSTAB (p < 0.001) were all important in controlling loss of mass of lysozyme over time. However, no significant difference was found when the samples were stored with and without the addition of GLB (p = 0.373).
No signal was seen on Western blots run with BIOSTAB, indicating that the enzyme stabilizer itself is not crossreactive with the WB procedure used in this experiment.
Previous preliminary work in our laboratories has shown that there is a substantial loss in lysozyme mass after extraction from SH lenses, particularly lotrafilcon-based materials, and subsequent processing (lyophilization, resuspension, and storage). Such loss in lysozyme mass has been previously reported by other groups looking at tears33 and saliva,34 but in these cases, the concentration of lysozyme was significantly higher than that typically found on lotrafilcon-based hydrogel lenses, which typically deposit <5 μg of lysozyme per lens.28, 31, 32 An alternative reason, not addressed in this article, for this loss in lysozyme mass is that lysozyme may undergo dimerization36 or aggregation,37 resulting in failure to be recognized by the antibody used in our WB assay. However, preliminary work in our laboratory suggests that dimerized lysozyme would be detected with the polyclonal antibody used in our assay. Thus, our goal was to devise a protocol to reduce the degree of lysozyme loss, because this would serve as a significant tool for many research areas in which the examination of small amounts of lysozyme, in either solution or on the surface of biomaterials, is important.
Lysozyme is a globular protein, which is relatively stable when compared with most other proteins found in tears. However, in the quantitation of lysozyme deposited on a contact lens (which, depending on the quantification method, may require a significant degree of initial processing such as extraction, lyophilization, resuspension, and storage), it is possible that the conformational state of any protein, including lysozyme, could be significantly altered. Altered conformation has significant implications if the quantitative technique being used uses an antibody to recognize the protein of interest (for example, WB or enzyme-linked immunosorbent assay) and may be more critical than if the protein is being quantified by a method that does not involve antibody recognition (for example, high-performance liquid chromatography).
The stability of proteins in solution has been a major concern for biotechnologists and the pharmaceutical industry. Several studies have been conducted, and it has been recognized that long-term stability of proteins can be improved by adding substances such as sugars (e.g., dextran,38–40 trehalose,41–43 sucrose43, 44), salts,45–49 and polyols such as sorbitol.50, 51 The current understanding of protein stabilization has been achieved by thermodynamic measurements of interactions and microenvironmental changes occurring on addition of a stabilizing compound and also through nuclear magnetic resonance spectroscopy, differential scanning calorimetry, and circular dichroism. It is believed that the stabilizing phenomenon is a complex one and no single mechanism is responsible for stabilization.
We set out to develop a protocol that would reduce the loss in lysozyme over time from an elute from a silicone hydrogel contact lens. The two potential protein stabilizers that we examined in this study were 0.9% saline and a proprietary product developed for protein stabilization (BIOSTAB Biomolecule Storage Solution). The presence of buffer or salt solution is believed to maintain the native conformational state of lysozyme.48 The stabilizing effects of salts have been attributed mainly to their ability to mask the protein of interest from the surrounding solvents. This exclusion of harsh solvents from the protein surface leads to “preferential hydration” of the protein or “preferential exclusion” of the additive from the protein surface, limiting their denaturing effect. BIOSTAB Biomolecule Storage Solution is a solution that is free of DNAse, RNAse, and proteases. This product is an aqueous solution, which contains a nonionic detergent and is nontoxic. The producers and distributors of the product claim that this product increases storability of biomolecules such as enzymes, antibodies, and DNA. Despite repeated attempts by us to obtain the exact chemical composition of the product, we have been unable to obtain any further information and thus are unable to ascertain what components were exactly responsible for imparting such a protective effect during our analysis. However, examination of Figs. 2 and 3 clearly demonstrate that this product has a marked influence in controlling the loss of lysozyme mass over storage time, with no apparent impact on its ability to be recognized by a suitable antibody.
The samples were tested by storing them with and without the addition of GLB to determine whether any of the components in the GLB was responsible for altering the structure of lysozyme. One of the major components in the GLB is glycerol (at a concentration of 30%). Glycerol itself has a potential stabilizing effect on protein molecules51, 52; however, we did not find any significant difference when the samples were stored with and without the addition of GLB.
This study was conducted on deposited lysozyme recovered from only one type of SH contact lens material. Further work must be undertaken to examine the impact of this protocol on other proteins and on proteins recovered from other types of SH lens materials.
We have optimized a procedure using an MRB, BIOSTAB Biomolecule Storage Solution, and storage at -70°C in which we have been able to reduce the percentage loss of lysozyme after extraction from lotrafilcon contact lenses from approximately 33% to <1%. This revised protocol will be of significant value for researchers interested in studying the deposition of proteins onto substrates in both ocular and nonocular research areas.
This study was conducted with funding provided by Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Foundation for Innovation (CFI), and Alcon Research Limited.
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