I n human eye pathology, much attention

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1 Rabbit tear proteins. I. Detection and quantitation of lysozyme in nonstimulated tears Benjamin Bonavida,* Alfred T. Sapse,** and Eli E. Sercarz Rabbit tear lysozyme was detected in low concentration in rabbit tears collected without any stimulation. Quantitation of rabbit tear lysozyme was found possible through the use of sensitive techniques that measure its specific enzymic activity on the bacterial cell walls of Micrococcus lysodeikticus. Rabbit tear lysozyme has an electrophoretic mobility identical to that of hen egg-white lysozyme on cellulose and acrylamide gel. The experimental use of rabbits for tear lysozyme studies is discussed. I n human eye pathology, much attention has been paid to lysozyme in tears and its variation in pathological conditions, 3-4 allergic manifestations, 5 and autoimmune diseases. 0 Recently, smog eye irritation has been correlated with a low tear lysozyme level 7 and workers around fumes s also have a reduced lysozyme concentration. In order to be able to study the effect of environmental conditions on tear lysozyme, a suitable experimental animal is a necessity. A favored subject in experimental eye investigation has been the rabbit, but heretofore it has been reported that lysozyme was absent from rabbit tears. - 2 From the Department of Bacteriology, University of California, Los Angeles, Calif This investigation was supported in part by a grant from the Arthritis Foundation of Southern California and by Grant No. NB from the United States Public Health Service, National Institute of Neurological Diseases and Blindness. "Recipient of United States Public Health Traineeship No. 5T0-GM '^Recipient of Public Health Service Special Fellowship No. F NB This study seeks to determine whether rabbit tears contain lysozyme, and consequently whether the rabbit can be used to study the environmental effect of different experimental pathological conditions on the tear lysozyme. In this report, lysozyme will be identified: () by its lyric activity on Micrococcus lysodeikticus, (2) by its electrophoretic mobility as compared with hen egg-white lysozyme, and (3) by the fact that lysozyme can be efficiently adsorbed by bentonite. Materials and methods Experimental white albino rabbits of different ages and sexes were chosen randomly to determine the lysozyme content of their tears. Standard Schirmer strips or microcapillary tubes were used for tear collection. When microcapillary tubes were employed for tear collection, care was taken to collect only the tears that were already present in the external canthus of the eyes under the lids. The amount usually collected from each eye was 5 to 0 Ail. When the Schirmer method was used, only the first few millimeters of the paper were allowed to moisten with tears. Either 2.5 fi\ tears or a 5 mm. 2 Schirmer strip was enough for assaying lysozyme. Protein determination. Protein concentration was determined by the Lowry 20 method and bo-

2 436 Bonavida, Sapse, and Sercarz Investigative Ophthalmology August 968 Table I. Quantitative determination of lysozyme in rabbit tears Rabbit No NRS Protein concentration (mg./ml. tears) Schirmer plate Lysozyme content (/Jig/ml Smolelis and Hartsell tears) Cellulose acetate electrophoresis After adsorption with bentonite vine serum albumin (BSA) was used as a standard of comparison. Cellulose acetate electrophoresis. Electrophoresis was performed on cellulose polyacetate strips with the use of the standard LKB apparatus model No. 50. The buffer used was 0.M sodium acetate, ph 4.7. The conditions of the run were 250 v., 0 Ma. for one hour. Protein bands were fixed and stained with Ponceau S that contained 5 per cent trichoroacetic acid. When a cellulose strip was used for localization of the lysozyme bands through its application on a Micrococcus plate (method to be discussed), the strip was cut longitudinally into two halves, one half of which was stained for reference. Lysozyme determination.* A. The Schirmer lysoplate method. This method consists of the determination of lysozyme in 2.5 fi\ tear samples which are either collected on Schirmer's paper strips or adsorbed to a 5 by 5 mm. square of paper. The papers are applied directly to the surface of agarose plates seeded with M. lysodeikticus cells. Plates are incubated at 37 C. for 24 hours. The diameters of the zones of lysis obtained are measured and the amount of lysozyme present in test samples is determined from a standard curve. The method has been described in more detail elsewhere. 9 B. The Smolelis and Hartsell method. 0 This method measures the decrease in optical density at 5400 A of a standard suspension of M. lysodeikticus after 0 minutes' incubation at 37 C. of the cell lysozyme mixture. C. Cellulose acetate electrophoresis method. After electrophoresis of rabbit tears on cellulose acetate strips, the strips were cut longitudinally into two halves, one of which was applied on the surface of an agarose layer seeded with M. lysodeikticus prepared in Petri dishes. 9 The plates were left for incubation at 37 C. for 24 hours. A of the methods described hereafter were enzymatic and used M. lysodeikticus as a substrate. The radii of the zones of lysis obtained surrounding the putative lysozyme bands were measured. The amounts of rabbit tear lysozyme present in the tear samples tested were extrapolated from a reference standard curve prepared with various concentrations of pure hen egg-white lysozyme (HEL). D. Acrylamide gel electrophoresis. Acrylamide gel electrophoresis was performed according to the method described by Reisfeld and colleagues, 3 for the separation of basic proteins, with a voltage of 320 v., 5 Ma. per tube, for 45 minutes. Staining was done with per cent Amido Schwartz for hour and destaining was done electrophoretically with 7 per cent acetic acid for several hours. E. Bentonite adsorption. Lysozyme has been found to be removable from serum by adsorption onto bentonite particles, 2 because of its basic ionic characteristic. The rabbit tear samples were mixed with a small quantity of dry bentonite and then centrifuged. The supernatant was tested for the presence of lysozyme by the Schirmer lysoplate method. Results Lysozyme Quantitation in Rabbit Tears. The concentrations of lysozyme in nonstimulated rabbit tears are summarized in Table I. Eight rabbits were tested which were chosen randomly. A. The Schirmer lysoplate method. In tears collected on Schirmer paper strips, the lysozyme concentration ranged between 60 and 70 jug per milliliter of tears with an average of 02 /xg per milliliter. A control normal rabbit serum has a lysozyme concentration of 30 /.ig per milliliter of serum (Table I).

3 ! ;! - : Volume 7 Number 4 Rabbit tear proteins. I z. / / :: i _ 20 I -I UJ 50 I : :. : E YV- r ; d : i, t I, : j/f : :! : - -i-!t " t j m ' ' i!! ]d I s : ; I.-.I -: i r r Mi i i! ;TI V i M : i..., ;! i! i : i i i!... :. :! iiiili i ;!-i" i i i : i I j ' ' i -i.ii! n.ii ; ; : j- (! i M:: hi:... i... j ' i' ; :+...LL: : i i j - ; ; i! liii i i I ' i RADIUS OF LYTIC ZONE (mm) Fig.. The drawing on the left side represents a Petri dish that has been seeded with a suspension of M. lysodeikticus in agarose. After electrophoresis, the cellulose strip was applied on the surface of tine plate and incubated at 37 C. for 24 hours. The lytic zone obtained, presumably around the lysozyme band, is visible. The dotted lines on the cellulose strip represent the protein bands obtained after staining of tine other half of tine strip. The radius of tine lytic zone is measured. On the right side, a standard curve was prepared with various concentrations of HEL, and the radius of the lytic zone obtained by tine cellulose acetate electrophoresis method was measured and plotted against the corresponding concentration. Note the linear relationship obtained. Equalizing the protein concentrations in tears and serum, we find that the lysozyme concentration in rabbit tears is about 4 times greater than in serum. The protein content of rabbit tears is greater than that of human tears; in the latter, the protein content is 7.2 mg. per milliliter of tears, whereas in rabbits it averages 2.6 mg. per milliliter. Human tear lysozyme (HTL) comprises 20 to 40 per cent of the total tear protein/ 7 whereas in rabbit tears, lysozyme comprises only per cent. B. The Smolelis and Hartsell method. 0 The results obtained with this method agree very closely with those obtained with the use of Schirmer lysoplate method (Table I). C. Cellulose acetate electrophoresis method. Rabbit tears upon electrophoresis on cellulose polyacetate strips give 6 to 7 bands. In only out of 8 cases were we able to visualize a stained protein band that corresponded in position to a control hen egg lysozyme (HEL) band. This rabbit had the highest lysozyme concentration in its tears (70 /xg per milliliter, rabbit No, 2). Following the electrophoresis of tears on cellulose acetate strips, the strips were cut longitudinally into two halves. One half was layered on a Micrococcus plate and left to incubate 24 horns; the other half of the strip was stained. The lysozyme concentrations of the tear samples were extrapolated from the standard curve shown in Fig. (Table I). The results with this assay show that: () the lysozyme content of each tear sample appears to be decreased relative to the Schirmer lysoplate method; and (2) the cathodal position of the lytic zone obtained from the tear samples corresponded to the electropho-

4 In vestigatioe Ophthalmology August Bonavida, Sapse, and Sercarz H R. EARS b HEL R TEARS HEL Fig. 2. Gel electrophoresis on acrylamide was performed according to the procedure of Reisfeld for the separation of basic proteins. The direction of electrophoresis is from top to the bottom, toward the cathode. Represented from right to left: a, samples of rabbit tears; b, hen egg lysozyme; c, a mixture of rabbit tears and hen egg lysozyme. Notice the presence of a lysozyme band in a and its resemblance in migration to HEL in b and c. retic position of HEL, despite the absence of an observable, stained band on the strip. D. Acrylamide gel electrophoresis. The normal pattern of rabbit tear proteins on acrylamide gel electrophoresis will be described elsewhere. Under conditions used for migration of basic proteins, rabbit serum gave many bands, whereas rabbit tears gave 8 bands, the most cathodal of which corresponded in migration to a sample of purified HEL (Fig. 2). When rabbit tears and HEL were mixed before layering the sample on the acrylamide tube, this most cathodal band was the only one which appeared more dense, since HEL staining was coincident with this band in rabbit tears. E. Bentonite adsorption. Since bentonite has been found to adsorb lysozyme completely,2 treatment of the rabbit tear sample with bentonite should result in the absence of any lysozyme activity. This was found to be the case, as is shown in Table I, where the assay used was the Schirmer lysoplate. Discussion The results obtained from these experiments indicate the presence of a protein component in rabbit tears which () has an enzymatic activity on Micrococcus lysodeikticus cell walls, (2) migrates electrophoretically toward the cathode to a position identical with that of hen egg lysozyme (HEL), and (3) is removable upon bentonite adsorption. These properties are also shared by all lysozymes so far studied3; thus the protein component presumably is a lysozyme. The presence of lysozyme in rabbit tears was sought by several investigators, none of whom were able to demonstrate it in nonstimulated rabbit tears. Goldsworthy and Florey showed the absence of lysozyme in rabbit tears using a method based on the reduction in turbidity of a suspension of Micrococcus lysodeikticus as a measure of the amount of lysozyme. Erickson and colleagues2 used a filter paper electrophoresis technique and noticed the absence of lysozyme in rabbit tears; their

5 Volume 7 Number 4 Rabbit tear proteins. I 439 results were confirmed by McDonald, Leopold, and Sery. 4 Kimura and colleagues 5 reported the presence of lysozyme in tears when the rabbit eyes had been infected or irritated. The lysozyme concentration in rabbit tears is very low compared to that of humans." ' l7 However, its concentration is greater than in the circulation. Its isolation and purification from rabbit tears in sufficient quantity for characterization is not feasible; however, Jolles ls has isolated and purified lysozyme from rabbit spleen. Its over-all amino acid composition is quite different from HEL 9 and human lysozymes. Since it showed a similar electrophoretic mobility as HEL, we can assume that rabbit tear lysozyme has a net positive charge similar to that of HEL. In the quantitation of rabbit tear lysozyme, it has been estimated that its specific enzymic activity is similar to HEL. In fact this was demonstrated with rabbit spleen lysozyme, TS which we assume has a similar enzymatic activity as rabbit tear lysozyme (RTL). We have used methods based upon enzymatic activity to demonstrate the presence of lysozyme in rabbit tears. Even though our assay systems were basically identical with those of other investigators, they do differ in their sensitivity. We have used a method of measuring lysozyme in a final concentration of less than 0 ng per milliliter of solution. 9 We have also shown that lysozyme detection solely by electrophoretic and staining techniques is not sensitive enough for detecting low levels of lysozyme. There exists a certain threshold below which paper electrophoresis techniques are not capable of detecting the relatively low level of lysozyme in rabbit tears. From our staining experiments on cellulose acetate strips after electrophoresis, we have shown that in 7 out of 8 cases it was impossible to detect the protein unless its enzymatic activity was utilized to localize its position on the strip. The presence of lysozyme in rabbit tears permits the use of this valuable experimental animal in studies of the tear proteins. For example, it has recently been shown that lysozyme levels in human subjects with smog eye irritation are markedly decreased. 7 Experiments under defined atmospheric conditions in rabbits may lead to an understanding of the causative agent(s) responsible for the alteration in the tear lysozyme level and possibly smog eye irritation. We wish to thank Mr. Solomon Bonavicla for his technical assistance. REFERENCES. Goldsworthy, N. E. 5 and Florey, N.: Some Properties of Mucus, with special reference to its antibacterial functions, Brit. J. Exper. Path. : 92, Erickson, O. F., Feeney, L., and McEwen, W. K.: Filter Paper Electrophoresis of Tears. II. Animal tears and the presence of a slow moving lysozyme, Arch. Ophth. 55: 800, McEwen, K., and Kimura, S. J.: Filter Paper Electrophoresis. I. Lysozyme and its correlation with keratoconjunctivitis sicca, Am. J. Ophth. 39: 200, Erickson, O. F.: The absence of lysozyme in Sjogren's syndrome, Stanford M. Bull. 3: 292, Erickson, O. F.: Allergic Etiology and Lysozyme Production, INVEST. OPHTH. 2: 98, Minton, R.: Paralimbal Ring Keratitis and Absence of Lysozyme in Lupus Erythematosus, Am. J. Ophth. 60: 532, Sapse, A. T., Bonavida, B., Stone, W., Jr., and Sercarz, E. E.: Human Tear Lysozyme. III. Preliminary study on lysozyme levels in subjects with smog eye irritation, Am. J. Ophth. July, Erickson, O., Hatlen, R., and Berg, M.: Industrial Tear Study, Am. J. Ophth. 47: 499, Bonavida, B., and Sapse, A. T.: Human Tear Lysozyme. II. A sensitive method for its quantitative determination making use of standard Schirmer strips, Am. J. Ophth. July, Smolelis, A. N., and Hartsell, S. E.: The Determination of Lysozyme, J. Bact. 58: 73, Reisfeld, R. A., Lewis, U. J., and Williams, D. E.: Disk electrophoresis of basic proteins and peptides on polyacrylamide gels, Nature 95: 28, Inai, S., Kishimoto, S., Hirao, F., and Takabashi, H.: Studies on serum lysozyme, presented at the Eleventh Meeting of Kansai Branch of the Society of Japanese Bacteriologists, Oct. 9, Jolles, P., Charlemagne, D. ; Petit, F. F.,

6 440 Bonavida, Sapse, and Sercarz Investigatioe Ophthalmology August 968 Marie, E., and Jolles, J.: Biochemie comparee des lysozymes, Bull. Soc. chim. biol. 47: 224, McDonald, P. R., Leopold, J. H., and Sery, T. W.: The Potential Value of Human Tear Patterns, Tr. Am. Ophth. Soc. 55: 49, McEwen, VV. K., Kimura, S. J., Feeney, M. L., and Li, T. G.: Lysozyme in Tears and in Leukemic Leukocytes, in The First International Symposium on Fleming's Lysozyme, Milan, 959, Abstracts of relations and communication, p Jolles, J., and Jolles, P.: Human Tear and Human Milk Lysozymes, Biochemistry 6: 4, Bonavida, B., Sapse, A. T., and Sercarz, E. E.: Human Tear Lysozyme. I. Purification, physicochemical and immunochemical characterization, J. Lab. & Clin. Med. 70: 95, Jolles, G., and Fromageot, C.: La proteine lysante II de la rate du lapin. II. Composition en acides amines, Biochem. et biophys. acta 4: 29, Canfield, R. E.: The Amino Acid Sequence of Egg-White Lysozyme, J. Biol. Chem. 238: 2698, Lowry, O. H., and Hunter, T. H.: The Determination of Serum Protein Concentration with a Gradient Tube, J. Biol. Chem. 59: 465, 945.