SUBBARAMAN, LAKSHMAN N. BSOptom, MSc; GLASIER, MARY-ANN MSc; SENCHYNA, MICHELLE PhD; JONES, LYNDON PhD, FCOptom, FAAO
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 This data (unpublished) indicates that this loss in mass is particularly problematic with lysozyme deposition on lotrafilcon lens materials.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
1. Bohnert JL, Horbett TA, Ratner BD, Royce FH. Adsorption of proteins from artificial tear solutions to contact lens materials. Invest Ophthalmol Vis Sci 1988;29:362–73.
2. Minarik L, Rapp J. Protein deposits on individual hydrophilic contact lenses: effects of water and ionicity. CLAO J 1989;15:185–8.
3. Hart DE, Tidsale RR, Sack RA. Origin and composition of lipid deposits on soft contact lenses. Ophthalmology 1986;93:495–503.
4. Jones L, Evans K, Sariri R, Franklin V, Tighe B. Lipid and protein deposition of N-vinyl pyrrolidone-containing group II and group IV frequent replacement contact lenses. CLAO J 1997;23:122–6.
5. Bontempo AR, Rapp J. Protein and lipid deposition onto hydrophilic contact lenses in vivo. CLAO J 2001;27:75–80.
6. Sack RA, Jones B, Antignani A, Libow R, Harvey H. Specificity and biological activity of the protein deposited on the hydrogel surface. Relationship of polymer structure to biofilm formation. Invest Ophthalmol Vis Sci 1987;28:842–9.
7. Castillo EJ, Koenig JL, Anderson JM, Jentoft N. Protein adsorption on soft contact lenses. III. Mucin. Biomaterials 1986;7:9–16.
8. Brennan NA, Coles M-LC. Deposits and symptomatology with soft contact lens wear. Int Contact Lens Clin 2000;27:75–100.
9. Gellatly KW, Brennan NA, Efron N. Visual decrement with deposit accumulation of HEMA contact lenses. Am J Optom Physiol Opt 1988;65:937–41.
10. Jones L, Franklin V, Evans K, Sariri R, Tighe B. Spoilation and clinical performance of monthly vs three monthly group II disposable contact lenses. Optom Vis Sci 1996;73:16–21.
11. Allansmith MR. Immunologic effects of extended-wear contact lenses. Ann Ophthalmol 1989;21:465–7, 74.
12. Porazinski AD, Donshik PC. Giant papillary conjunctivitis in frequent replacement contact lens wearers: a retrospective study. CLAO J 1999;25:142–7.
13. Allansmith MR, Korb DR, Greiner JV, Henriquez AS, Simon MA, Finnemore VM. Giant papillary conjunctivitis in contact lens wearers. Am J Ophthalmol 1977;83:697–708.
14. Donshik PC. Contact lens chemistry and giant papillary conjunctivitis. Eye Contact Lens 2003;29:S37–9; discussion S57–9, S192–4.
15. Aswad MI, John T, Barza M, Kenyon K, Baum J. Bacterial adherence to extended wear soft contact lenses. Ophthalmology 1990;97:296–302.
16. Butrus SI, Klotz SA, Misra RP. The adherence of Pseudomonas aeruginosa to soft contact lenses. Ophthalmology 1987;94:1310–4.
17. Butrus SI, Klotz SA. Contact lens surface deposits increase the adhesion of Pseudomonas aeruginosa. Curr Eye Res 1990;9:717–24.
18. Wedler FC. Analysis of biomaterials deposited on soft contact lenses. J Biomed Mater Res 1977;11:525–35.
19. Wedler FC, Illman BL, Horensky DS, Mowrey-McKee M. Analysis of protein and mucin components deposited on hydrophilic contact lenses. Clin Exp Optom 1987;70:59–68.
20. Franklin V, Bright A, Pearce E, Tighe B. Hydrogel lens spoilation, part 5: tear proteins and proteinaceous films. Optician 1992;204:16–26.
21. Keith DJ, Christensen MT, Barry JR, Stein JM. Determination of the lysozyme deposit curve in soft contact lenses. Eye Contact Lens 2003;29:79–82.
22. Castillo EJ, Koenig JL, Anderson JM, Lo J. Protein adsorption on hydrogels. II. Reversible and irreversible interactions between lysozyme and soft contact lens surfaces. Biomaterials 1985;6:338–45.
23. Garrett Q, Chatelier RC, Griesser HJ, Milthorpe BK. Effect of charged groups on the adsorption and penetration of proteins onto and into carboxymethylated poly(HEMA) hydrogels. Biomaterials 1998;19:2175–86.
24. Garrett Q, Garrett RW, Milthorpe BK. Lysozyme sorption in hydrogel contact lenses. Invest Ophthalmol Vis Sci 1999;40:897–903.
25. Janssen PT, van Bijsterveld OP. Origin and biosynthesis of human tear fluid proteins. Invest Ophthalmol Vis Sci 1983;24:623–30.
26. Maissa C, Franklin V, Guillon M, Tighe B. Influence of contact lens material surface characteristics and replacement frequency on protein and lipid deposition. Optom Vis Sci 1998;75:697–705.
27. Alvord L, Court J, Davis T, Morgan CF, Schindhelm K, Vogt J, Winterton L. Oxygen permeability of a new type of high Dk soft contact lens material. Optom Vis Sci 1998;75:30–6.
28. McNally J, McKenney C. A clinical look at a silicone hydrogel extended wear lens. Contact Lens Spectrum 2002;17:38–41.
29. Morgan CF, Brennan NA, Alvord L. Comparison of the coulometric and polarographic measurement of a high-Dk hydrogel. Optom Vis Sci 2001;78:19–29.
30. Jones L. Modern contact lens materials: a clinical performance update. Contact Lens Spectrum 2002;17:24–35.
31. Jones L, Senchyna M, Glasier MA, Schickler J, Forbes I, Louie D, May C. Lysozyme and lipid deposition on silicone hydrogel contact lens materials. Eye Contact Lens 2003;29:S75–9; discussion S83–4, S192–4.
32. Senchyna M, Jones L, Louie D, May C, Forbes I, Glasier MA. Quantitative and conformational characterization of lysozyme deposited on balafilcon and etafilcon contact lens materials. Curr Eye Res 2004;28:25–36.
33. Sitaramamma T, Shivaji S, Rao GN. Effect of storage on protein concentration of tear samples. Curr Eye Res 1998;17:1027–35.
34. Ng V, Koh D, Fu Q, Chia SE. Effects of storage time on stability of salivary immunoglobulin A and lysozyme. Clin Chim Acta 2003;338:131–4.
35. Keith D, Hong B, Christensen M. A novel procedure for the extraction of protein deposits from soft hydrophilic contact lenses for analysis. Curr Eye Res 1997;16:503–10.
36. Scott G, Mowrey-McKee M. Dimerization of tear lysozyme on hydrophilic contact lens polymers. Curr Eye Res 1996;15:461–6.
37. Gu Z, Zhu X, Ni S, Su Z, Zhou HM. Conformational changes of lysozyme refolding intermediates and implications for aggregation and renaturation. Int J Biochem Cell Biol 2004;36:795–805.
38. Sasahara K, McPhie P, Minton AP. Effect of dextran on protein stability and conformation attributed to macromolecular crowding. J Mol Biol 2003;326:1227–37.
39. Allison SD, Chang B, Randolph TW, Carpenter JF. Hydrogen bonding between sugar and protein is responsible for inhibition of dehydration-induced protein unfolding. Arch Biochem Biophys 1999;365:289–98.
40. Liao YH, Brown MB, Quader A, Martin GP. Protective mechanism of stabilizing excipients against dehydration in the freeze-drying of proteins. Pharm Res 2002;19:1854–61.
41. Kaushik JK, Bhat R. Why is trehalose an exceptional protein stabilizer? An analysis of the thermal stability of proteins in the presence of the compatible osmolyte trehalose. J Biol Chem 2003;278:26458–65.
42. Sola-Penna M, Meyer-Fernandes JR. Stabilization against thermal inactivation promoted by sugars on enzyme structure and function: why is trehalose more effective than other sugars? Arch Biochem Biophys 1998;360:10–4.
43. Ueda T, Nagata M, Imoto T. Aggregation and chemical reaction in hen lysozyme caused by heating at pH 6 are depressed by osmolytes, sucrose and trehalose. J Biochem (Tokyo) 2001;130:491–6.
44. Remmele RL Jr, Stushnoff C, Carpenter JF. Real-time in situ monitoring of lysozyme during lyophilization using infrared spectroscopy: dehydration stress in the presence of sucrose. Pharm Res 1997;14:1548–55.
45. Takano K, Tsuchimori K, Yamagata Y, Yutani K. Contribution of salt bridges near the surface of a protein to the conformational stability. Biochemistry 2000;39:12375–81.
46. Arakawa T, Bhat R, Timasheff SN. Preferential interactions determine protein solubility in three-component solutions: the MgCl2 system. Biochemistry 1990;29:1914–23.
47. Arakawa T, Bhat R, Timasheff SN. Why preferential hydration does not always stabilize the native structure of globular proteins. Biochemistry 1990;29:1924–31.
48. Malzert A, Boury F, Renard D, Robert P, Proust JE, Benoit JP. Influence of some formulation parameters on lysozyme adsorption and on its stability in solution. Int J Pharm 2002;242:405–9.
49. Perez C, Griebenow K. Effect of salts on lysozyme stability at the water-oil interface and upon encapsulation in poly(lactic-co-glycolic) acid microspheres. Biotechnol Bioeng 2003;82:825–32.
50. Wimmer H, Olsson M, Petersen MT, Hatti-Kaul R, Peterson SB, Muller N. Towards a molecular level understanding of protein stabilization: the interaction between lysozyme and sorbitol. J Biotechnol 1997;55:85–100.
51. Grandori R, Matecko I, Mayr P, Muller N. Probing protein stabilization by glycerol using electrospray mass spectrometry. J Mass Spectrom 2001;36:918–22.
52. Knubovets T, Osterhout JJ, Connolly PJ, Klibanov AM. Structure, thermostability, and conformational flexibility of hen egg-white lysozyme dissolved in glycerol. Proc Natl Acad Sci U S A 1999;96:1262–7.