Optometry & Vision Science:
Stabilization of Lysozyme Mass Extracted From Lotrafilcon Silicone Hydrogel Contact Lenses
SUBBARAMAN, LAKSHMAN N. BSOptom, MSc; GLASIER, MARY-ANN MSc; SENCHYNA, MICHELLE PhD; JONES, LYNDON PhD, FCOptom, FAAO
Centre for Contact Lens Research, School of Optometry, University of Waterloo, Waterloo, Ontario, Canada
This work was presented as a poster at ARVO 2004, Ft. Lauderdale, Florida.
Received October 18, 2004; accepted December 20, 2004.
Purpose. Lysozyme deposits extracted from lotrafilcon silicone hydrogel (SH) contact lens materials demonstrate a loss in total mass as a function of storage time when assessed by Western blotting. This loss represents a potential source of error when quantifying total lysozyme deposition on SH lenses. The purpose of this study was to devise a method whereby lysozyme mass would be preserved over time to allow for its accurate quantitation after its removal from SH lenses.
Methods. Lysozyme deposits from 12 human worn lotrafilcon lenses were extracted using a 50:50 mixture of 0.2% trifluoroacetic acid and acetonitrile. Extracts were lyophilized to dryness, then resuspended in either reconstitution buffer (10 mM Tris-HCl, 1 mM EDTA) or modified reconstitution buffer (reconstitution buffer + 0.9% saline). BIOSTAB Biomolecule Storage Solution (Sigma-Aldrich) was added to one half of the samples from each buffer group. One microliter of each of the samples was immediately subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting, whereas the remaining volume was aliquoted and stored at -20°C or -70°C and subjected to the same procedures after 48 h of storage. Comparison of lysozyme band intensity in stored vs. fresh samples enabled calculation of percentage mass loss of lysozyme.
Results. Samples stored at -20°C in reconstitution buffer with no BIOSTAB demonstrated a 33% loss in mass over 48 h of storage. Identical samples stored at -70°C in modified reconstitution buffer with BIOSTAB added demonstrated <1% loss in mass. Statistical analysis 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 over time.
Conclusion. We have optimized a procedure whereby the extracted mass of lysozyme deposits found on lotrafilcon SH lenses can be preserved, thus enabling accurate quantitation after extraction and resuspension.
One of the major problems with hydrophilic contact lenses is that they are susceptible to spoilage from constituents of the tear film, which include a wide variety of proteins, lipids, and mucins.1–8 At extreme levels of buildup, these deposits are associated with diminished visual acuity9 and a feeling of dryness and discomfort.10 Deposits can ultimately lead to more serious clinical conditions such as hypersensitivity reactions and giant papillary conjunctivitis.11–14 Moreover, these deposits potentially increase the risk of bacterial attachment by providing a solid substrate and shelter.15–17
Tear film proteins frequently detected on hydrogel contact lenses include lysozyme, lactoferrin, and albumin,18–20 and among these, lysozyme has been the most widely studied.6, 21–24 Lysozyme is a compact globular protein molecule with a molar mass of 14.5 kDa. It is a bacteriolytic enzyme that is derived from the acinar and ductal epithelial cells of both main and accessory lacrimal glands.25 It is a positively charged molecule and this, coupled with its small size, results in its increased adsorption onto negatively charged substrates such as U.S. Food and Drug Administration group IV contact lens materials.2, 6, 23, 24, 26
The newly introduced silicone hydrogel (SH) contact lenses have significantly increased oxygen transmission as a result of the incorporation of Siloxane groups.27–29 The incorporation of silicone results in an increased degree of hydrophobicity, which results in increased lipid deposition compared with other nonsilicone-containing materials.30 However, these lens materials do deposit extremely low levels of protein compared with conventional hydrogel lenses, with typical levels being in the <20 μg/lens range.28, 31, 32
In two recent studies undertaken on tear and salivary samples, it was demonstrated that a reduction in lysozyme quantity occurs as a function of storage time.33, 34 To date, no study has been conducted on the effect of storage on lysozyme deposits that have been extracted from contact lens materials. Preliminary results in our laboratory have demonstrated that lysozyme deposits extracted from SH contact lens materials also demonstrate a loss in total mass after lyophilization and resuspension as a function of storage time when assessed by Western blotting (WB). This data (unpublished) indicates that this loss in mass is particularly problematic with lysozyme deposition on lotrafilcon lens materials. This loss represents a potential source of error when quantifying total lysozyme deposition. Moreover, the amount of lysozyme extracted from SH materials is very low, such that even a minimal loss would be significant in the interpretation of the total amount of lysozyme deposited. Thus, the purpose of this work was to devise a method whereby lysozyme mass would be preserved over time and would be compatible with a previously published WB procedure optimized in our laboratory.32
MATERIALS AND METHODS
The sample variables examined in this study included the presence or absence of gel-loading buffer (GLB), temperature (-20°C and -70°C), composition of two reconstitution buffers (RB and modified RB [MRB]) and the presence or absence of a biomolecule stabilizing agent (BIOSTAB). These six conditions with the two buffers (see Fig. 1) were examined systematically as described subsequently. Each trial was conducted in triplicate, resulting in a total of 60 samples being measured.
Reagents and Materials
All PhastSystem precast gels, buffer strips, well combs, filter paper, and ECL-Plus kits were purchased from Amersham Pharmacia Biotech (Baie d'Urfe, QC, Canada). Immuno-Blot polyvinylidene difluoride (PVDF) membrane was purchased from Bio-Rad Laboratories (Mississauga, ON, Canada). Polyclonal rabbit antihuman lysozyme was purchased from Cedarlane Laboratories (Hornby, ON, Canada), and goat antirabbit IgG-HRP was purchased from Sigma (St. Louis, MO). Human lysozyme (neutrophil) was purchased from Calbiochem (La Jolla, CA). A product developed specifically for stabilizing proteins and enzymes (BioStab Biomolecule Storage Solution [BIOSTAB]) was purchased from Sigma-Aldrich. All other reagents purchased were analytical grade and obtained from Sigma (St. Louis, MO).
Protein Deposit Extraction From Contact Lenses
Twelve lotrafilcon (Focus Night&Day; CIBA Vision, Atlanta, GA) SH contact lenses were collected after 4 weeks of daily wear use, during which subjects had disinfected the lenses with AOSept (CIBA Vision). Lenses were aseptically collected using nonpowdered surgical gloves and placed in individual glass vials containing 1.5 mL extraction solution consisting of a 50:50 mix of 0.2% trifluoroacetic acid and acetonitrile (ACN/TFA).35 The lenses were incubated in darkness at room temperature for 24 h. Two 0.6-mL aliquots of ACN/TFA were transferred to sterile Eppendorf tubes and lyophilized to dryness in a Savant Speed Vac (Halbrook, NY). Dried protein pellets were stored at -70°C before reconstitution.
Sample Processing After Extraction
Fig. 1 describes the sample processing after resuspension of the lyophilized sample extracts. Four 600-μL aliquots (2 by 600 μL from the right eye lens and 2 by 600 μL from the left eye lens of the same subject) of lyophilized lens extracts were taken, and three of them were resuspended in 20 μL of either a standard RB (10 mM Tris-HCl, 1 mM EDTA, pH 12) or a MRB (10 mM Tris-HCl, 1 mM EDTA, 0.9% saline, pH 12). Three of these 20-μL aliquots were pooled to total 60 μL, and this volume was added to the fourth 600-μL aliquot of lyophilized Focus Night&Day lens extract.
Addition of Enzyme Stabilizer
A total of 0.5 μL of the initial stock was taken and checked for neutrality using pH paper (Hydrion Papers; Micro Essential Laboratory, Brooklyn, NY). Once neutrality was confirmed, 4 μL of the sample was added to 10 polypropylene sample tubes (Axygen MAXYMum Recovery; Axygen Scientific, Inc, Union City, CA). A total of 2.5 μL of BIOSTAB was added to five samples and the same quantity of MilliQ water was added to the remaining five samples, which acted as the control group.
Addition of Gel-Loading Buffer
Six of the 10 samples were diluted with gel-loading buffer (GLB; 5% SDS, 100 mM Tris, pH 7.4, 30% glycerol, 1 mM EDTA, 0.02% bromophenol blue). The remaining four samples were stored under various conditions without GLB.
One set of samples were run without storage (fresh) and further samples were stored for 48 h under various conditions (Fig. 1).
Electrophoresis and Immunoblotting
Once prepared, samples were subjected to SDS-polyacrylamide gel electrophoresis followed by WB to PVDF membranes using the PhastSystem (Amersham-Pharmacia Biotech) as described previously.32 Lysozyme was identified using a rabbit antihuman lysozyme polyclonal antibody (Calbiochem), followed by a peroxidase conjugated goat antirabbit secondary antibody (Sigma-Aldrich). Individual standard curves of purified human neutrophil lysozyme (Calbiochem) were run on each gel to facilitate regression analysis. Immunoreactivity was visualized by incubating with ECL-Plus chemiluminescent substrate (Amersham-Pharmacia Biotech). Optical densities of the resulting bands were quantified from digitized images created with a Molecular Dynamics Storm 840 Imager using ImageQuant 5.1. Comparison of lysozyme band intensity in stored vs. fresh samples enabled calculation of percentage mass loss of lysozyme.
Testing for BioStab Crossreactivity in Western Blotting
To test if the enzyme stabilizer itself had any possible crossreactivity during the WB procedure, BIOSTAB in conjunction with buffer alone was subjected to SDS-polyacrylamide gel electrophoresis and WB to PVDF membranes using the PhastSystem, as described previously.
Table 1 and Fig. 2 show the percentage of lysozyme loss when the lyophilized sample extracts were resuspended in the “standard” reconstitution buffer, stored with and without the addition of BIOSTAB, GLB, and under the two storage temperatures (-20°C and -70°C). The addition of BIOSTAB clearly reduces the amount of lysozyme loss.
Table 2 and Fig. 3 show the percentage of lysozyme loss when the lyophilized sample extracts were resuspended in the “modified” reconstitution buffer, stored with and without the addition of BIOSTAB, GLB, and under the two storage conditions (-20°C and -70°C). The addition of BIOSTAB clearly reduces the degree of lysozyme loss.
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.
Centre for Contact Lens Research
School of Optometry
University of Waterloo
200 University Avenue
West Waterloo, Ontario
N2L 3G1 Canada
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