Two-dimensional electrophoresis of human tear proteins
Electrophoresis 1992, 13, 379-382
379
Seijiro Mii‘ Kazuyuki Nakamura’ Kazusuke Takeo’ Shinji Kurimoto’
Analysis of human tear proteins by two-dimensional electrophoresis
’ Department of Ophthalmology,
Human tear proteins in the conjunctival sac were separated on the basis of the differences in their isoelectric points and molecular weights using micro two-dimensional electrophoresis combined with immunoblotting. The two-dimensional electrophoretic patterns of tear proteins from patients with conjunctivitis were compared with those from normal individuals. We also measured integrated intensities of seven protein spots, lactoferrin (LF),albumin and five specific tear proteins (STP), to examine differences in the amounts of these proteins in tears from normal individuals of different sexes. In the tears from patients with conjunctivitis, secretory immunoglobulin A (IgA), LF and STP spots were stained more weakly, whereas the albumin spot was stained more strongly as compared with those from normal individuals. Furthermore, haptoglobin and IgG spots appeared in the tears from patients with conjunctivitis. These were more prominent in the tears from patients with severe conjunctivitis. There were significant differences in the amounts of LF and two kinds of STPs in the different sexes. The amounts of these proteins were larger in females.
’ The First Department of
Biochemistry, Yamaguchi University School of Medicine, Ube
1 Introduction
2 Materials and methods
Human tear proteins have been analyzed using various kinds of electrophoresis. Recently Coyle et al. [l]analyzed human tear proteins, using isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separately. Since there were several proteins which had similar isoelectric points (PI) or molecular weights (M,) in human tears, we separated human tear proteins on the basis of the differences in their pZs and M,s using micro two-dimensional electrophoresis combined with immunoblotting.
2.1 Sample preparation
Inada et al. [2,3] and Yamanouchi et al. [4] showed electrophoretic patterns of tear proteins from patients with various diseases using crossed immunoelectrophoresis and IEF, respectively. No one has shown two-dimensional electrophoretic patterns of tear proteins from patients with conjunctivitis. Sullivan et al. [5] reported that the concentration of the free secretory component was different in tears of male and female rats, although no protein having different concentrations in tears from human males and females has been reported. We compared the two-dimensional electrophoretic patterns of tear proteins from patients with conjunctivitis with those from normal individuals. Furthermore, the integrated intensities of three protein spots, namely lactoferrin (LF) and two kinds of specific tear proteins (STP), were measured to show sex differences in tear proteins from normal individuals.
~
Correspondence: Dr. Seijiro Mii, Department of Ophthalmology,Yamaguchi University School of Medicine, 1144 Kogushi, Ube, 755 Japan
Abbrevations: Alb, albumin; CBB, Coomassie Brilliant Blue; Hp, haptoglobin; IEF, isoelectric focusing; IgG, immunoglobulin G; LF, lactoferrin; M,, molecular weight; NC,§nitrocellulose; PAGE,polyacrylamide gel eieckTophoTesis; PI,isoelectric point; sIgA, secretory immunoglobulin A; SDS, sodium dodecyl sulfate; STP, specific tear protein; Tris, tris(hydroxymethy1)aminomethane Q VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
Tear samples were collected from 36 normal individuals (17 males and 19 females, ranging in age from 20 to 30 years old, who had no abnormality in their eyes except refractive errors and that they wore contact lenses) and from 15 patients with infectious conjunctivitis. Tears were obtained as follows: 30 pL sterilized physiological saline was dropped into the inferior conjunctival sac, and the inferior lacrimal punctum was oppressed gently. After one mild blinking, one end of a glass hematocrit capillary (BLU . TIP, plain, Monoject Scientific, St. Louis, MO, USA) was put near the inner canthus to collect approximately 20 pL of tears. Then the capillary containing tears was immediately stored at -20°C until use. After thawing, tear samples were centrifuged for 10 min at 12000 X g to yield a clear supernatant for subjecting to electrophoresis.
2.2 Protein assay The microassay method with a nitrocellulose (NC) membrane (0.22 pm pore size GS,Nihon Millipore Kogyo,Yonezawa, Japan) and “BIO DOT” microfiltration apparatus (Bio-Rad Laboratories, Richmond, CA, USA)[6] was used to measure protein concentrations of tear samples. Three pL of tear samples were spotted on an NC membrane and stained with Ponceau 3R (Wako Pure Chemical Industries, Osaka, Japan). After drying, the NC membrane was made translucent with liquid paraffin (Katayama Chemical, Osaka, Japan) to measure optical densities at 500 nm using a densitometer (Densitron PAN-802, Jookoo, Tokyo, Japan). Bovine serum albumin (Sigma, ST. Louis, MO, USA) was used as a standard protein.
2.3 Two-dimensional electrophoresis IEF with capillary polyacrylamide gels (4%, 1.3 X 35 mm) and SDS-PAGE with gradient slab gels (4-20°/o, 40X38X1 mm) were carried out in the first and in the second dimension, respectively, in a modified version of the method de0173-0838/92/0606-0379 $3.50+.28/0
380
Elrrrrophore.si.s 1992, 13, 379-382
S. Mii er a!.
scribed by Manabe et ul. [7] with twin gels, one for protein staining and another for immunostaining. Tear samples containing 20 pg and 10 pg of protein were prepared for protein staining and for immunostaining, respectively. In the first dimension, Ampholine (2.5% w/v, pH 3.5-10: pH 3.5-5 in a 4:l ratio; Pharmacia LKB Biotechnology, Bromma, Sweden) was used as a carrier ampholyte. Precision Disposable Micropipettes (100 pL; Drummond Scientific Company,Broomall,PA, USA) were used as glass capillaries for gel casting. I E F was carried out at a constant current of 0.1 niA/gel until the voltage increased to 300V and then at a constant voltage of 300Vfor20 min in an ice water bath. In the second dimension, SDS-PAGE was carried out at a constant current of 2.5 mA/gel at room temperature for 3 h. Saccharose, acrylamide and N,N-methylenebisacrylamide(Bis) were purchased from Seikagaku Kogyo (Tokyo, Japan). SDS and N,N,N,N-tetramethylethylenediamine (TEMED) were purchased from Nacalai Tesque (Kyoto, Japan). Ammonium peroxydisulfate, sodium hydroxide, phosporic acid, glycine, Broniophenol Blue, methanol and acetic acid were purchased from Katayama Chemical. Tris(hydroxymethy1)aminomethane (Tris) was purchased from Sigma.
2.4 Protein staining and immunoblotting After SDS-PAGE in the second dimension was completed, tear proteins in one gel were stained with Coomassie Brilliant Blue (CBB) R-250 (Nacalai Tesque) [7] and tear proteins in another gel were transferred to an NC membrane using a senii-dry electroblotter (SM17556, Sartorius, Germany). Blotting was carried out at a constant voltage of 6 V at room temperature for 2 h. Anode buffer 1 contained 0.3 M Tris and 20% v/v methanol, pH 10.4,and anode buffer 2 contained 25 mM Tris and 20% methanol, pH 10.4. The cathode buffer contained 25 mM Tris, 20% methanol and 40 mM 6-amino-n-caproic acid (Nacalai Tesque), pH 9.4. After blotting was completed, the NC membrane was subjected to immunostaining. The NC membrane was twice washed with Tris-buffered saline (TBS) for 30min and blocked with 5% w/v skim milk (Morinaga Milk,Tokyo, Japan) in TBS for 1h. After the NC membrane was washed with TBS for 30 s, the membrane was incubated in goat antihuman whole serum antiserum (Cappel, Westchester, PA, USA) as the first antibody in a 500-fold dilution, with TBS containing 1O/o skim milk, for 3 h. After the NC membrane was washed four times with TBS containing. 0.05% v/v Tween 20 (Katayama Chemical) for IOmin, the membrane was incubated in a peroxidase-conjugated rabbit anti-goat IgG antibody (Cappel) solution as the second antibody in a 2000-fold dilution, with TBS containing 1% skim milk, for 2 h. After the NC membrane was washed four times with TBS containing 0.05 O/o Tween 20 for 10 min, the color was developed in a solution containing 0.05 O/o w/v 4-chloro-lnaphthol (Sigma), 16.7% methanol and 0.015% v/v hydrogen peroxide (Mitsubishi Gas Chemical, Tokyo, Japan) for 10min at room temperature.
2.5 Measurement of integrated intensities of protein spots After protein staining with CBB R-250, integrated intensities of seven protein spots (LF, albumin and five STPs) in tears of normal individuals were measured using a Bio Image 80s (MilliGen/Biosearch Division of Millipore,
Tokyo, Japan). Statistical analysis was carried out with Mann-Whitney approval to examine sex differences in those proteins.
3 Results 3.1 Tear protein concentrations The protein concentrations of tear samples were 2.0 2 1.4 g/L(mean k SD) in the normal individuals (1.4 f 1.1 g/Lin normal males and 2.3 f 1.5 g/L in normal females) and 4.0 f2.9 g/Lin the patients with conjunctivits (4.0 21 2.7 g/Lin 11 patients with mild conjunctivitis and 4.2 2r 3.7 g/L in four patients with severe conjunctivitis). The protein concentrations of tear samples were significantly higher 0)< 0.05) in the patients with conjunctivitis as compared with those in the normal individuals, although there were no significant differences in the protein concentrations of tear samples between the normal males and females, or between the patients with mild conjunctivitis and the patients with severe conjunctivitis. 3.2 Two-dimensional electrophoretic patterns of tear proteins from normal individuals By staining with CBB R-250 (Fig. l), proteins identified according to their pls and A4,s are secretory immunoglobulin A (sIgA) at the position of high Mr > 100000, LF and albumin at M, 50000-100000,and lysozyme at Mr< 50000.Five spos (1-9, with the exception of the lysozyme spot, could be detected in the range of M, lower than that of albumin. Four of the five spots (2-5) could be detected at the position ofpH 5 and M, 15000.Thefive spots which could be detected by protein staining with CBB R-250 were not stained by immunostaining with the anti-human whole serum antiserum (Fig. 2). These spots corresponded to the proteins specific for human tears reported by Bonavida et ul. [8], Gachon et ul. [9] and Inada et ul. [lO].Thus,we referred to these five spots as STP -1, -2, -3, -4 and -5, respectively.
D H 3.5 I-
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Two-dimensional electrophoresis of human tear proteins
Electrophoresis 1992, 13,379-382
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Figure 2. Immunostaining of tear proteins (slgA and Alb) from a normal individual with anti-human whole serum antiserum.
3.3 Two-dimensional electrophoretic patterns of tear proteins from patients with conjunctivitis
P H 3. 5
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In the tears from patients with mild conjunctivitis (Fig. 3A), sIgA, LF and STPs spots were stained more weakly, whereas the albumin spot was stained more strongly, as compared with those from normal individuals. Furthermore, haptoglobin (Hp) and immunoglobulin G (IgG) spots appeared in the tears from patients with mild conjunctivitis. They were even more prominent in the tears from patients with severe conjunctivitis (Fig. 3B).
- 66
3.4 Tear proteins with sex differences There were significant sex differences in the integrated intensities of LF (p < O.Ol), STP-2(p < 0.05) and STP-S(p < 0.05) spots (Fig. 4). The mean values of integrated intensities in arbitrary units of the protein spots were 66.3 for LF, 55.5 for STP-2, and 11.3 for STP-5 in females, these being higher than those of 35.9 for LF, 38.7 for STP-2, and 7.4 for STP-5 in males. However, other protein spots (albumin, STP-1,-3, and-4) did not show significant sex differences in the integrated intensities.
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Figure 3. Two-dimensional electrophoretic patterns of tear proteins from patients with conjunctivitis. (A): Tears from a patient with mild conjunctivitis. (B) Tears from a patient with severe conjunctivitis.The samples contain 20 vg ofprotein.The gels are stained with CBB R-250. Proteins as in Fig. 1; Hp, haptoglobin.
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Figure 4. Sex differences in the amounts of tear proteins. Integrated intensities of (A) LF, (B) STP-2 and (C) STP-5, measured using a Bio Image as described in Section 2.5. Tears were collected from 17 normal males and 19 normal females. The bars indicate the mean values of integrated intensities of the protein spots, expressed in arbitrary units.
382
Electrophoresrj 1992. 13. 379-382
S MII "1 a/
4 Discussion 4.1 Tear protein assay The measurement of protein concentrations was carried out to adjust the amount of proteins in the tear samples for electrophoresis.Lowry's method [ l l ] has been used forprotein determination of body fluids, however, 20 pL of tear samples were necessary for this method when the samples contained low concentrations of proteins. Therefore, the microassay method was used because it required only 3 pL of tear samples for protein measurement.
4.2 Tear proteins from normal individuals Inada et a / . [12] reported that in the tear samples from normal individuals,Hp was detected when tears were collected by stimulating eyes with onions, and serum proteins were barely detected when tears were collected by diluting them with physiological saline. Stuchell et a / . [13] reported that IgC was detected in tear samples from normal individuals when tears were collected by absorbing them into a Schirmer filter paper strip. We collected tears by diluting them with sterilized physiological saline. Hp and IgG could not be detected in tear samples from normal individuals by immunostaining with anti-human whole serum antiserum (Fig.2).This method did not increase the contamination by serum proteins of the tear samples from normal individuals during collection. It is important to select the method for collecting tears with minimized contamination by serum proteins.Albumin and four STP spots (STP-2,-3,-4,and -5) had similar pls (about pH 5 ) , and lysozyme and the four STP spots had similar M,s of 15000. We could separate these protein spots on the basis of the differences in their pls and M,s by means of high resolution two-dimensional electrophoresis (Fig. 1).
-
4.3 Tear proteins from patients with conjunctivitis Inada et a / . [2] reported that sIgA increased in the tears from patients with severe keratoconjunctivitis. However, in our study, sIgA decreased in the tears from patients with conjunctivitis. The decrease of sIgA was even more prominent in the tears from patients with severe conjunctivitis. This may be due to the increase in the amount of serum proteins in the tears from patients with conjunctivitis. The amount ofHp, IgC and albumin increased in the tears from patients with conjunctivitis. Particularly the amount of Hp increased with an increase in the severity of conjunctivitis (Fig. 3B). The intensity of Hp spots may be a good indicator for an estimation of the severity of conjunctivitis.
4.4 Sex differences in the amounmts of tear proteins The differences in the amounts of STP-2 and STP-5 in the different sexes were statistically significant (Fig. 4B, C).The changes in the integrated intensities of these spots were not due to the changes in the amounts of other major proteins because other STP spots (STP-1, -3, and -4) did not change their integrated intensities with the changes of STP-
2 and STP-5 in the same tear samples. The STPs were also separated by crossed immunoelectrophoresis [2], however, their structures and functions are still unknown. The amounts of STP-2 and STP-5 were significantly higher in females in our results.This may indicate that the induction of STP-2 and STP-5 are regulated by gender hormones. Suzuki et al. [14] reported that there was no significant sex difference in the concentration of LF in the tears from normal individuals estimated by the enzyme-linked immunosorbent assay (ELISA) method when tears were collected by stimulating eyes with onions. In our study, the amounts of LF were significantly higher in females (Fig. 4A). Yamashita et al. [15] reported that estrogen induced LF in uterine epithelial cells. It is supposed that LF in lacrimal gland cells is also induced by estrogen. LF has an important role in the nonspecific host defense system [16,17]; there may therefore be a sex difference in the ability to defend against infection at the anterior segment of the eyes of normal individuals. We are grateful to Dr. ltsuko Sugihara,forher assistance with the statistical analysis, to Dr. Tomoko Miyazaki f o r her assistance in sample preparation and to Mr. Motonori Akahori f o r his technical assistance in the measurement of integrated intensities of the protein spots separated by two-dimensional electrophoresis. Received December 17, 1991
References Coyle, P. K., Sibony, P. A . and Johnson, C., Invest. Ophthalmol. Vis. Sci. 1989, 30, 1872-1878.
Inada,K.,Baba,H. and Okamura, R.,ActaSoc. Ophthalmol. J p n . 1983, 87, 1187-1191. Inada, K., Baba, H . and Okamura, R., Jpn. J. Ophrhalmol. 1985.29, 212-221. Yamanouchi,K.,Kohno,K.and Honmura, S., J p n . Rev. Clin. Ophthalmol. 1987, 81, 316-320. Sullivan, D. A , , Bloch, K. J . and Allansmith, M. R., J. Immunol. 1984, 132, 1130-1135. Nakamura, K., Tanaka, T., Kuwahara, A . and Takeo, K., Anal. Biochem. 1985, 148,311-319. Manabe, T. and Okuyama, T., in: Hirai, H., Oda, T., Matsubashi, N. and Shimao, K. (Eds.). Denkieidojikkenho Bunkodo,Tokyo 1989, pp. 3 15-331. Bonavida, B., Sapse, A. T. and Sercarz, E. E., Nature 1969, 221, 375-376. Gachon,A. M.,Lambin, P. and Dastugue, B., Ophthalmic. Res. 1980, 12, 277-285. Inada, K., Jpn. J. Ophthalmol. 1984, 28, 315-330. Lowry, 0.H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., J . B i d . Chem. 1951, 193,265-275. Inada,K.,Baba,H. and Okamura. R.,ActaSoc. Ophthalmd. J p n . 1983, 87,482-489. Stuchell, R. N., Feldman, J . J., Farris, R. L. and Mandel, I. D., Invest. Ophthalmol. Vis. Sci. 1984, 25, 374-317. Suzuki,Y., Nakao, T., Matsuda, S., Nakagawa,T. and Niida, Y., Folia Ophthalmol. Jpn. 1986, 37, 659-663. Yamashita, S., Newbold, R. R., McLachlan, J. A. and Korach, K. S., Endociynology 1990, 127,2456-2463. Arnold, R. R., Russell, J. E., Champion, W. J., Brewer, M. and Gauthier, J. J., Infect. Immun. 1982, 35, 792-799. Brock, J. H., Pickering, M. G., McDowall, M . C. and Deacon, A. G., Inject. Immun. 1983, 40, 453-459.
Electrophoresis 1992, 13, 379-382
379
Seijiro Mii‘ Kazuyuki Nakamura’ Kazusuke Takeo’ Shinji Kurimoto’
Analysis of human tear proteins by two-dimensional electrophoresis
’ Department of Ophthalmology,
Human tear proteins in the conjunctival sac were separated on the basis of the differences in their isoelectric points and molecular weights using micro two-dimensional electrophoresis combined with immunoblotting. The two-dimensional electrophoretic patterns of tear proteins from patients with conjunctivitis were compared with those from normal individuals. We also measured integrated intensities of seven protein spots, lactoferrin (LF),albumin and five specific tear proteins (STP), to examine differences in the amounts of these proteins in tears from normal individuals of different sexes. In the tears from patients with conjunctivitis, secretory immunoglobulin A (IgA), LF and STP spots were stained more weakly, whereas the albumin spot was stained more strongly as compared with those from normal individuals. Furthermore, haptoglobin and IgG spots appeared in the tears from patients with conjunctivitis. These were more prominent in the tears from patients with severe conjunctivitis. There were significant differences in the amounts of LF and two kinds of STPs in the different sexes. The amounts of these proteins were larger in females.
’ The First Department of
Biochemistry, Yamaguchi University School of Medicine, Ube
1 Introduction
2 Materials and methods
Human tear proteins have been analyzed using various kinds of electrophoresis. Recently Coyle et al. [l]analyzed human tear proteins, using isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separately. Since there were several proteins which had similar isoelectric points (PI) or molecular weights (M,) in human tears, we separated human tear proteins on the basis of the differences in their pZs and M,s using micro two-dimensional electrophoresis combined with immunoblotting.
2.1 Sample preparation
Inada et al. [2,3] and Yamanouchi et al. [4] showed electrophoretic patterns of tear proteins from patients with various diseases using crossed immunoelectrophoresis and IEF, respectively. No one has shown two-dimensional electrophoretic patterns of tear proteins from patients with conjunctivitis. Sullivan et al. [5] reported that the concentration of the free secretory component was different in tears of male and female rats, although no protein having different concentrations in tears from human males and females has been reported. We compared the two-dimensional electrophoretic patterns of tear proteins from patients with conjunctivitis with those from normal individuals. Furthermore, the integrated intensities of three protein spots, namely lactoferrin (LF) and two kinds of specific tear proteins (STP), were measured to show sex differences in tear proteins from normal individuals.
~
Correspondence: Dr. Seijiro Mii, Department of Ophthalmology,Yamaguchi University School of Medicine, 1144 Kogushi, Ube, 755 Japan
Abbrevations: Alb, albumin; CBB, Coomassie Brilliant Blue; Hp, haptoglobin; IEF, isoelectric focusing; IgG, immunoglobulin G; LF, lactoferrin; M,, molecular weight; NC,§nitrocellulose; PAGE,polyacrylamide gel eieckTophoTesis; PI,isoelectric point; sIgA, secretory immunoglobulin A; SDS, sodium dodecyl sulfate; STP, specific tear protein; Tris, tris(hydroxymethy1)aminomethane Q VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
Tear samples were collected from 36 normal individuals (17 males and 19 females, ranging in age from 20 to 30 years old, who had no abnormality in their eyes except refractive errors and that they wore contact lenses) and from 15 patients with infectious conjunctivitis. Tears were obtained as follows: 30 pL sterilized physiological saline was dropped into the inferior conjunctival sac, and the inferior lacrimal punctum was oppressed gently. After one mild blinking, one end of a glass hematocrit capillary (BLU . TIP, plain, Monoject Scientific, St. Louis, MO, USA) was put near the inner canthus to collect approximately 20 pL of tears. Then the capillary containing tears was immediately stored at -20°C until use. After thawing, tear samples were centrifuged for 10 min at 12000 X g to yield a clear supernatant for subjecting to electrophoresis.
2.2 Protein assay The microassay method with a nitrocellulose (NC) membrane (0.22 pm pore size GS,Nihon Millipore Kogyo,Yonezawa, Japan) and “BIO DOT” microfiltration apparatus (Bio-Rad Laboratories, Richmond, CA, USA)[6] was used to measure protein concentrations of tear samples. Three pL of tear samples were spotted on an NC membrane and stained with Ponceau 3R (Wako Pure Chemical Industries, Osaka, Japan). After drying, the NC membrane was made translucent with liquid paraffin (Katayama Chemical, Osaka, Japan) to measure optical densities at 500 nm using a densitometer (Densitron PAN-802, Jookoo, Tokyo, Japan). Bovine serum albumin (Sigma, ST. Louis, MO, USA) was used as a standard protein.
2.3 Two-dimensional electrophoresis IEF with capillary polyacrylamide gels (4%, 1.3 X 35 mm) and SDS-PAGE with gradient slab gels (4-20°/o, 40X38X1 mm) were carried out in the first and in the second dimension, respectively, in a modified version of the method de0173-0838/92/0606-0379 $3.50+.28/0
380
Elrrrrophore.si.s 1992, 13, 379-382
S. Mii er a!.
scribed by Manabe et ul. [7] with twin gels, one for protein staining and another for immunostaining. Tear samples containing 20 pg and 10 pg of protein were prepared for protein staining and for immunostaining, respectively. In the first dimension, Ampholine (2.5% w/v, pH 3.5-10: pH 3.5-5 in a 4:l ratio; Pharmacia LKB Biotechnology, Bromma, Sweden) was used as a carrier ampholyte. Precision Disposable Micropipettes (100 pL; Drummond Scientific Company,Broomall,PA, USA) were used as glass capillaries for gel casting. I E F was carried out at a constant current of 0.1 niA/gel until the voltage increased to 300V and then at a constant voltage of 300Vfor20 min in an ice water bath. In the second dimension, SDS-PAGE was carried out at a constant current of 2.5 mA/gel at room temperature for 3 h. Saccharose, acrylamide and N,N-methylenebisacrylamide(Bis) were purchased from Seikagaku Kogyo (Tokyo, Japan). SDS and N,N,N,N-tetramethylethylenediamine (TEMED) were purchased from Nacalai Tesque (Kyoto, Japan). Ammonium peroxydisulfate, sodium hydroxide, phosporic acid, glycine, Broniophenol Blue, methanol and acetic acid were purchased from Katayama Chemical. Tris(hydroxymethy1)aminomethane (Tris) was purchased from Sigma.
2.4 Protein staining and immunoblotting After SDS-PAGE in the second dimension was completed, tear proteins in one gel were stained with Coomassie Brilliant Blue (CBB) R-250 (Nacalai Tesque) [7] and tear proteins in another gel were transferred to an NC membrane using a senii-dry electroblotter (SM17556, Sartorius, Germany). Blotting was carried out at a constant voltage of 6 V at room temperature for 2 h. Anode buffer 1 contained 0.3 M Tris and 20% v/v methanol, pH 10.4,and anode buffer 2 contained 25 mM Tris and 20% methanol, pH 10.4. The cathode buffer contained 25 mM Tris, 20% methanol and 40 mM 6-amino-n-caproic acid (Nacalai Tesque), pH 9.4. After blotting was completed, the NC membrane was subjected to immunostaining. The NC membrane was twice washed with Tris-buffered saline (TBS) for 30min and blocked with 5% w/v skim milk (Morinaga Milk,Tokyo, Japan) in TBS for 1h. After the NC membrane was washed with TBS for 30 s, the membrane was incubated in goat antihuman whole serum antiserum (Cappel, Westchester, PA, USA) as the first antibody in a 500-fold dilution, with TBS containing 1O/o skim milk, for 3 h. After the NC membrane was washed four times with TBS containing. 0.05% v/v Tween 20 (Katayama Chemical) for IOmin, the membrane was incubated in a peroxidase-conjugated rabbit anti-goat IgG antibody (Cappel) solution as the second antibody in a 2000-fold dilution, with TBS containing 1% skim milk, for 2 h. After the NC membrane was washed four times with TBS containing 0.05 O/o Tween 20 for 10 min, the color was developed in a solution containing 0.05 O/o w/v 4-chloro-lnaphthol (Sigma), 16.7% methanol and 0.015% v/v hydrogen peroxide (Mitsubishi Gas Chemical, Tokyo, Japan) for 10min at room temperature.
2.5 Measurement of integrated intensities of protein spots After protein staining with CBB R-250, integrated intensities of seven protein spots (LF, albumin and five STPs) in tears of normal individuals were measured using a Bio Image 80s (MilliGen/Biosearch Division of Millipore,
Tokyo, Japan). Statistical analysis was carried out with Mann-Whitney approval to examine sex differences in those proteins.
3 Results 3.1 Tear protein concentrations The protein concentrations of tear samples were 2.0 2 1.4 g/L(mean k SD) in the normal individuals (1.4 f 1.1 g/Lin normal males and 2.3 f 1.5 g/L in normal females) and 4.0 f2.9 g/Lin the patients with conjunctivits (4.0 21 2.7 g/Lin 11 patients with mild conjunctivitis and 4.2 2r 3.7 g/L in four patients with severe conjunctivitis). The protein concentrations of tear samples were significantly higher 0)< 0.05) in the patients with conjunctivitis as compared with those in the normal individuals, although there were no significant differences in the protein concentrations of tear samples between the normal males and females, or between the patients with mild conjunctivitis and the patients with severe conjunctivitis. 3.2 Two-dimensional electrophoretic patterns of tear proteins from normal individuals By staining with CBB R-250 (Fig. l), proteins identified according to their pls and A4,s are secretory immunoglobulin A (sIgA) at the position of high Mr > 100000, LF and albumin at M, 50000-100000,and lysozyme at Mr< 50000.Five spos (1-9, with the exception of the lysozyme spot, could be detected in the range of M, lower than that of albumin. Four of the five spots (2-5) could be detected at the position ofpH 5 and M, 15000.Thefive spots which could be detected by protein staining with CBB R-250 were not stained by immunostaining with the anti-human whole serum antiserum (Fig. 2). These spots corresponded to the proteins specific for human tears reported by Bonavida et ul. [8], Gachon et ul. [9] and Inada et ul. [lO].Thus,we referred to these five spots as STP -1, -2, -3, -4 and -5, respectively.
D H 3.5 I-
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D H 3.5
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381
Two-dimensional electrophoresis of human tear proteins
Electrophoresis 1992, 13,379-382
5
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Figure 2. Immunostaining of tear proteins (slgA and Alb) from a normal individual with anti-human whole serum antiserum.
3.3 Two-dimensional electrophoretic patterns of tear proteins from patients with conjunctivitis
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In the tears from patients with mild conjunctivitis (Fig. 3A), sIgA, LF and STPs spots were stained more weakly, whereas the albumin spot was stained more strongly, as compared with those from normal individuals. Furthermore, haptoglobin (Hp) and immunoglobulin G (IgG) spots appeared in the tears from patients with mild conjunctivitis. They were even more prominent in the tears from patients with severe conjunctivitis (Fig. 3B).
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3.4 Tear proteins with sex differences There were significant sex differences in the integrated intensities of LF (p < O.Ol), STP-2(p < 0.05) and STP-S(p < 0.05) spots (Fig. 4). The mean values of integrated intensities in arbitrary units of the protein spots were 66.3 for LF, 55.5 for STP-2, and 11.3 for STP-5 in females, these being higher than those of 35.9 for LF, 38.7 for STP-2, and 7.4 for STP-5 in males. However, other protein spots (albumin, STP-1,-3, and-4) did not show significant sex differences in the integrated intensities.
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Figure 4. Sex differences in the amounts of tear proteins. Integrated intensities of (A) LF, (B) STP-2 and (C) STP-5, measured using a Bio Image as described in Section 2.5. Tears were collected from 17 normal males and 19 normal females. The bars indicate the mean values of integrated intensities of the protein spots, expressed in arbitrary units.
382
Electrophoresrj 1992. 13. 379-382
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4 Discussion 4.1 Tear protein assay The measurement of protein concentrations was carried out to adjust the amount of proteins in the tear samples for electrophoresis.Lowry's method [ l l ] has been used forprotein determination of body fluids, however, 20 pL of tear samples were necessary for this method when the samples contained low concentrations of proteins. Therefore, the microassay method was used because it required only 3 pL of tear samples for protein measurement.
4.2 Tear proteins from normal individuals Inada et a / . [12] reported that in the tear samples from normal individuals,Hp was detected when tears were collected by stimulating eyes with onions, and serum proteins were barely detected when tears were collected by diluting them with physiological saline. Stuchell et a / . [13] reported that IgC was detected in tear samples from normal individuals when tears were collected by absorbing them into a Schirmer filter paper strip. We collected tears by diluting them with sterilized physiological saline. Hp and IgG could not be detected in tear samples from normal individuals by immunostaining with anti-human whole serum antiserum (Fig.2).This method did not increase the contamination by serum proteins of the tear samples from normal individuals during collection. It is important to select the method for collecting tears with minimized contamination by serum proteins.Albumin and four STP spots (STP-2,-3,-4,and -5) had similar pls (about pH 5 ) , and lysozyme and the four STP spots had similar M,s of 15000. We could separate these protein spots on the basis of the differences in their pls and M,s by means of high resolution two-dimensional electrophoresis (Fig. 1).
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4.3 Tear proteins from patients with conjunctivitis Inada et a / . [2] reported that sIgA increased in the tears from patients with severe keratoconjunctivitis. However, in our study, sIgA decreased in the tears from patients with conjunctivitis. The decrease of sIgA was even more prominent in the tears from patients with severe conjunctivitis. This may be due to the increase in the amount of serum proteins in the tears from patients with conjunctivitis. The amount ofHp, IgC and albumin increased in the tears from patients with conjunctivitis. Particularly the amount of Hp increased with an increase in the severity of conjunctivitis (Fig. 3B). The intensity of Hp spots may be a good indicator for an estimation of the severity of conjunctivitis.
4.4 Sex differences in the amounmts of tear proteins The differences in the amounts of STP-2 and STP-5 in the different sexes were statistically significant (Fig. 4B, C).The changes in the integrated intensities of these spots were not due to the changes in the amounts of other major proteins because other STP spots (STP-1, -3, and -4) did not change their integrated intensities with the changes of STP-
2 and STP-5 in the same tear samples. The STPs were also separated by crossed immunoelectrophoresis [2], however, their structures and functions are still unknown. The amounts of STP-2 and STP-5 were significantly higher in females in our results.This may indicate that the induction of STP-2 and STP-5 are regulated by gender hormones. Suzuki et al. [14] reported that there was no significant sex difference in the concentration of LF in the tears from normal individuals estimated by the enzyme-linked immunosorbent assay (ELISA) method when tears were collected by stimulating eyes with onions. In our study, the amounts of LF were significantly higher in females (Fig. 4A). Yamashita et al. [15] reported that estrogen induced LF in uterine epithelial cells. It is supposed that LF in lacrimal gland cells is also induced by estrogen. LF has an important role in the nonspecific host defense system [16,17]; there may therefore be a sex difference in the ability to defend against infection at the anterior segment of the eyes of normal individuals. We are grateful to Dr. ltsuko Sugihara,forher assistance with the statistical analysis, to Dr. Tomoko Miyazaki f o r her assistance in sample preparation and to Mr. Motonori Akahori f o r his technical assistance in the measurement of integrated intensities of the protein spots separated by two-dimensional electrophoresis. Received December 17, 1991
References Coyle, P. K., Sibony, P. A . and Johnson, C., Invest. Ophthalmol. Vis. Sci. 1989, 30, 1872-1878.
Inada,K.,Baba,H. and Okamura, R.,ActaSoc. Ophthalmol. J p n . 1983, 87, 1187-1191. Inada, K., Baba, H . and Okamura, R., Jpn. J. Ophrhalmol. 1985.29, 212-221. Yamanouchi,K.,Kohno,K.and Honmura, S., J p n . Rev. Clin. Ophthalmol. 1987, 81, 316-320. Sullivan, D. A , , Bloch, K. J . and Allansmith, M. R., J. Immunol. 1984, 132, 1130-1135. Nakamura, K., Tanaka, T., Kuwahara, A . and Takeo, K., Anal. Biochem. 1985, 148,311-319. Manabe, T. and Okuyama, T., in: Hirai, H., Oda, T., Matsubashi, N. and Shimao, K. (Eds.). Denkieidojikkenho Bunkodo,Tokyo 1989, pp. 3 15-331. Bonavida, B., Sapse, A. T. and Sercarz, E. E., Nature 1969, 221, 375-376. Gachon,A. M.,Lambin, P. and Dastugue, B., Ophthalmic. Res. 1980, 12, 277-285. Inada, K., Jpn. J. Ophthalmol. 1984, 28, 315-330. Lowry, 0.H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., J . B i d . Chem. 1951, 193,265-275. Inada,K.,Baba,H. and Okamura. R.,ActaSoc. Ophthalmd. J p n . 1983, 87,482-489. Stuchell, R. N., Feldman, J . J., Farris, R. L. and Mandel, I. D., Invest. Ophthalmol. Vis. Sci. 1984, 25, 374-317. Suzuki,Y., Nakao, T., Matsuda, S., Nakagawa,T. and Niida, Y., Folia Ophthalmol. Jpn. 1986, 37, 659-663. Yamashita, S., Newbold, R. R., McLachlan, J. A. and Korach, K. S., Endociynology 1990, 127,2456-2463. Arnold, R. R., Russell, J. E., Champion, W. J., Brewer, M. and Gauthier, J. J., Infect. Immun. 1982, 35, 792-799. Brock, J. H., Pickering, M. G., McDowall, M . C. and Deacon, A. G., Inject. Immun. 1983, 40, 453-459.
