Current Eve Rksearch
SHORT COMMUNICATION Volume I 1 number 12 1992, 1239-1243
Protein analysis of monkey aqueous humor
Paul Russell and David L.Epstein'
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
National Eye Institute, National Institutes of Health, Bethesda, MD and 'Department of Ophthalmology, Duke University, Durham, NC, USA
ABSTRACT Aqueous humor from individual eyes of young monkeys (Macaca mulatta) was analyzed by gel exclusion chromatography, and one- and two-dimensional gel electrophoresis. The data indicated that in young normal monkey the main peak of protein eluting from gel exclusion columns was observed around 80,000 daltons. There was a small amount of heavy molecular weight material eluting from these columns with an apparent molecular size of greater than 500,000 daltons, but very little material smaller than 40,000 daltons. By one- and two-dimensional gel electrophoresis, the major polypeptide components in the monkey aqueous were similar to those reported for the human aqueous with polypeptides at around 170, 130, 110, 80, 67, 60, 42, 34, 28, 25, 22, 16, and 14 kilodaltons (kD). The monkey aqueous humor also contains the protease inhibitor cystatin.
guidelines on the use of animals in research. Because of the constraints of the vaccine testing, the animals were first exsanguinated, and immediately after death, a paracentesis of the aqueous humor was done. The paracenteses were done between 8 and 10 AM. A 25 gauge needle on a tuberculin syringe was used with the entry site just anterior to the limbus. The upward-facing bevel of the needle was observed during the removal of the aqueous. Aqueous humor (approximately 100p1) was obtained from each eye, and the individual samples were frozen at -7OOC. Seventy-six samples were used in this study. The protein content of each of the aqueous humor samples was determined using the Bradford method (7).
The aqueous humor of the vertebrate eye plays an essential role in the homeostasis of the tissues in the anterior part of the eye. The composition of the aqueous may play a role in the pathogenesis of cataract or glaucoma and also in wound healing (1-6). Surprisingly little information is available analyzing aqueous humor by gel
Bovine serum albumin was used as a standard. Four of the aqueous samples with a minimum of three aliquots from each were used exclusively for protein measurement. The mean of these four samples was 6.53 mg/dl f 1.77. The other 68 samples were measured using one aliquot of
indication of the apparent native sizes of the proteins in
either 10 or 20 p1. The mean of the 68 other samples was 6.18 mg/dl f 2.38. These values are closer to those reported for human (4) and for other non-human primates
solution. An earlier report on the composition of the
(6) than the value for Rhesus previously reported (3). One
aqueous humor of the Rhesus monkey (Macaca mulatta)
possible reason for the discrepancy in the values may be
suggested that several constituents were very similar to
the use of the different techniques for protein
those reported in human aqueous (3). We have recently
determination. Just as in human, the ascorbate in the
had the opportunity to obtain a number of Rhesus monkey
monkey aqueous humor can interfere with some protein
aqueous specimens, and have extended previous
quantitative methods. For a standard sample of lOmg/dl
observations to individual aqueous samples.
in lmh4 ascorbate, a Lowry protein determination (8) gave
exclusion chromatography. This analysis gives an
Monkeys (Macaca mulatta), aged two to three years,
an inflated protein content of 30.6mg/dl. No interference
were used by the vaccine testing service of the Bureau of
of ascorbate was noted with the Bradford method (data not
Biologics. The monkeys were normal animals. The tests
shown).
were performed in accordance with NIH institutional
High performance liquid chromatography (HPLC)
Received on August 10, 1992; accepted on November 9, 1992
0 Oxford University Press
1239
Current Eye Research kD
-200 -97
-68
-20 -14 0
1 1
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
wR
I\
0 Z
Size (kD)
I 135
F 1 2 3 4 5 6 7 8 91011
1
I
I
67
43
20
Figure 1. The elution pattern of a single sample of monkey aqueous humor eluted from a Superdex 75 column. The polypeptides, eluted from the column, were precipitated in trichloroacetic acid and run on SDS PAGE. The silver
stained gel pattern of the polypeptides in these fractions is shown in the inset. The calibration of the column using protein standards is shown below the figure.
was run using a Superdex 75 column or a Superose 12
little material eluted after 40kD. Polypeptides in the
column (Pharmacia-LKB,Piscataway, NJ) with a flow rate
fractions eluted from the column were precipitated in 10%
of 0.25 ml/min. A variable wave length monitor (LKB
trichloroacetic acid (TCA) by adding a tenth volume of
2151, Pharmacia-LKB) was set at 280nm with a full scale
100%TCA to each fraction. The tubes were cooled to 4OC for a minimum of 30 minutes and then centrifuged at
absorbance of 0.005. Fractions of 500~1were collected. Four different HPLC buffer systems were used. Two
14,000 xg for 20 minutes. The protein pellets were
buffer systems used l O O m M NaCl with either 50mM Tris-
solubilized in 5% sodium dodecylsulfate (SDS) in 62.5mM
HC1 (pH 7.1) or 50mM HEPES (pH 7.1). Both of these buffer systems gave identical results. The other two buffer
Tris-HC1 (pH 8.8) with 5% 0-mercaptoethanol. Typically, each fraction was solubilized in 2p1 of the SDS buffer. The
systems utilized 50 mM Tris-HC1, 100 mM KC1, 1 mh4
sample were analyzed with one-dimensional
EDTA, and 10 mM P-mercaptoethanol at pH values of either 7.1 or 7.4. These two buffer systems gave identical
polyacrylamide gel electrophoresis (PAGE) using the
results to the ones without a reducing agent or EDTA.
were stained with silver as previously reported with the
Superdex 75 chromatography on a 3Opl sample from one eye with a protein content of 5.2 mg/dl revealed a
exception that 5% citric acid was substituted for the 10%
small peak of absorbing material with a relatively large
only unresolved streaks of stain in the first fractions off the
size above about 200kD (Figure 1). There was a separation
column, but some of the heavier molecular weight
of two main absorbing peaks around 130 and 80kD. Very
polypeptides could be resolved beginning with the material
1240
PhastSystem (Pharmacia-LKB). The 12.5%resolving gels
acetic acid in the development solutions (9). There were
Current Eye Research BASIC
ACIDIC
li
kD 9743 -
20-
I A\ FRACT
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
SAMPLE A
E
A 1
1
1
1
1
1
1
1
5
FRACTION 1 I
Size (kD)
11 12 13 14 15 1617 11121314151617 SAMPLE B
1
1
1
1
1
1
I
2000 230
1
1
1
15
10
1
1
20
I
I
I
158
67
25
Figure 2. Su erose 12 column chromatographs of 501-11of a single s a m p i of monkey aqueous with a protein content of 5.8 mg/dl (A) and the two-dimensional gel electrophoresis pattern of the sample (left inset). The aqueous from the fellow eye (4.7mg/dl) was evaporated to dryness,
rehydrated, and then loaded onto the same column (B). Polypeptides precipitated from the fractions of sample B were run on SDS PAGE and are shown by the silver stained gel (right inset). The calibration of the column using protein standards is shown below the figure.
in fraction 4. Faint polypeptide bands around 170, 130 and
aqueous from the fellow eye (B)(4.7mg/dl) that was first
llOkD could be detected. There was a band at 67kD in
evaporated to dryness in a Speed Vac Concentrator
fraction 4 which may have been disulfide linked albumin.
(Savant, Farmingdale, NY) and then solubilized in H,O.
Fraction 5 and 6 showed the main band at 67kD and other
Drying was used to determine if this would adversely
bands around 80 and 60kD; additional polypeptides with
affect the sample. Concentration of the specimens was
smaller molecular weights were present in this fraction.
essential in order to run the two-dimensional
Two or three bands around 35kD as well as bands at 28,
electrophoresis. The chromatogram revealed a small
25,22,16, and 14kdD were observed. Later fractions
amount (4% of the total absorbance) of material eluting in
eluting from the column did not contain very much
the heavy molecular size range at approximately 500kD.
staining material on the SDS gel. Analyses of other
The chromatograph of the rehydrated sample B showed a
individual samples or even pooled samples on this column
somewhat larger amount of material eluting in this range.
gave similar results (n=8).
SDS gel analysis of these fractions revealed only an
To resolve the proteins with larger sizes, multiple
undiscernible smear of stain from the top of the gel down
samples (n=24) were also analyzed with the Superose 12
to about 67kD. The increased amount of material in the
column. The pattern of a sample (A)(5.8mg/dl) run on this
fractions near the start of elution in the samples that had
column (Figure 2) can be compared to the pattern of the
been dehydrated was always small but somewhat variable.
1241
Current Eye Research
I\
kD
E .02
68
-
rn'
14
-
h
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
5 .O I
1L
+ "
I
m
a
S
Figure 3. Elution pattern of pooled monkey aqueous humor from a Superose 12 column and dot blot (below) showing the immunoreactive cystatin eluted from the column primarily in fractions 14 with a weak positive reaction observed in fraction 15. (S) human saliva; positive control. (B) bovine serum albumin; negative control. Dot blots
were done on nitrocellulose. The seam after fraction 13 is the result of joining sequential strips of the dot blot together. (Inset) Silver stained SDS PAGE (I) and western blot (11) of an aqueous sample incubated with antibodies to cystatin. The positions of the molecular weight markers are shown.
Use of elution buffers which contained P-mercaptoethanol had no apparent effect on the elution profiles (data not
similar apparent molecular weights but various isoelectric
shown). The increase of material at this size range may
Several of the polypeptides below 60kD appear to have
represent aggregated protein that could not be sufficiently
very acidic isoelectric points and can be seen near the edge
dissociated during rehydration. However, concentration of
of the gel. Thus, both gel exclusion chromatography and
individual specimens by calibrated filter units such as the
two-dimensional analysis can be done with a single
Centricon-10 units (Amicon, Lexington, MA) resulted in a
aqueous specimen.
significant loss of material from individual samples. There
points may be modified proteins, perhaps glycoproteins.
Cystatin, a 14kD inhibitor of thiol protease, is found
was insufficient material recovered after concentration with
in relatively high quantities in tears and saliva (10).
the Centricon filters to enable further study.
Western blots were done using the protocol of Western
For two-dimensional analysis, an aliquot of sample
Lights (Tropix, Waltham, MA) with the primary antibody
A was concentrated by speedvac and rehydrated in one
diluted 1500. Five individual samples were run on SDS
tenth volume of 8M urea and 2% nonidet P40. The
PAGE and cystatin was detected in all specimens (Figure
sample was applied to the urea isoelectric focusing gel (pH
3). To determine where this small protein was eluting
3-9) and subsequently, the resolved proteins were run on a
from the Superose 12 column, a pooled aliquot of six
12.5% SDS gel (9). The two-dimensional analysis showed
the large proportion of the staining material in the monkey
aqueous samples was used. Dot blots of loop1 of each fraction were incubated with the antiserum for cystatin.
aqueous that was present at 67kD (inset, Figure 2). Several
Immunoreactivity was detected in fractions 14 and 15 in
individual polypeptides can be seen as well as "trains" of
the main peak of absorbance.
polypeptides. These groupings of staining material with
1242
A most interesting finding is the minimal amount of
Current Eye Research lower molecular weight material that i s apparently free in
Supported by grant NIH-NEI RO1-01894 and a grant from
the aqueous. It could be that the sizes of the aggregates
National Glaucoma Research, a Division of the American
that are filtered through the trabecular meshwork are
Health Assistance Foundation, Rockville, Maryland to
tightly controlled. Small hydrophobic proteins could
D.L.E.
potentially interact with the constituents of the trabecular meshwork and lead to the obstruction in flow of the
CORRESPONDING AUTHOR
aqueous (11). The current results would indicate that the
Paul Russell, Bldg. 6 Rm. 228, National Institutes of
bulk of the protein material that must pass through the
Health, Bethesda, MD 20892
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
trabecular meshwork has an apparent native size range between 200 and 40kD. A recent report (12) suggested that the plasma proteins diffuse into the anterior chamber through the anterior iris. If this model is correct, the data presented here indicate a possible role for albumin. Polypeptides having low molecular weights eluted from columns with the albumin fraction. These polypeptides could be aggregating, or they could be associating with albumin. The latter possibility is a very intriguing one since it would suggest that albumin may play an essential role in clearing these components through the trabecular meshwork. Protein-protein interactions within the trabecular meshwork would be lessened if these small proteins were first associated with albumin. This hypothesis would be consistent with the diffusion of the plasma in an area just prior to the outflow pathway. Furthermore, it suggests a previously unappreciated and essential role for albumin in the maintenance of the trabecular meshwork. The data show that the protein content of the Rhesus aqueous humor is nearer to the human value than the higher value previously reported. The results suggest that there are two major protein peaks that elute from gel filtration columns of monkey aqueous. These peaks have sizes of approximately 130 and 80kD and there is little protein material eluting in the size range below 40kD. The polypeptide composition of the Rhesus monkey aqueous appears to be quite similar to the human. ACKNOWLEDGEMENTS We wish to thank Mr. John Cogan of the Bureau of
REFERENCES 1. De Berardinis, E., Tieri, O., Polzella, A., and Iuglio, N. (1965) The chemical composition of the human aqueous humour in normal and pathological conditions. Exp. Eye Res. & 179-186. 2. Sandberg, H.O. and Closs, 0. (1979) The alpha and gamma crystallin content in aqueous humor of eyes with clear lenses and with cataracts. Exp. Eye Res.-82 601-610. 3. Gaasterland, D.E., Pederson, J.E., MacLellan, H.M., and Reddy, V.N. (1979) Rhesus monkey aqueous humor composition and a primate ocular perfusate. Invest. Ophthalmol. Vis. Sci. -8l 1139-1150. 4. Tripathi, R.C., Millard, C.B., and Tripathi, B.J. (1989) Protein composition of human aqueous humor: SDS PAGE analysis of surgical and post-mortem samples. Exp. Eye Res. 48, 117-130. 5. Jampel, H.D., Roche, N., Stark, W.J., and Roberts, A.B. (1990) Transformin growth factor-p in human aqueous humor. Curr. Eye 8es. 3 963-969. 6. Jampel, H.D., Brown, A., Roberts, A., Koya, P., and Quigley, H. (1992) Effect of paracentesis upon the blood-aqueous barrier of cynomolgus monkeys. Invest. Ophthalmol. Vis. Sci. -3 165-171. 7. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principles of protein-dye binding. Anal. Biochem. -27 248-254. 8. Peterson, G.L. (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. € 3 - 346-356. 9. Russell, P. and Yamada, T. (1990) Increased sensitivity of two-dimensional gel electrophoresis using PhastSystem. BioTechniques, 5 422-424. 10. Al-Hashimi, I., Dickinson, D.F, and Levine, M.J. (1988) Purification, molecular cloning, and sequencing of salivary cystatin SA-I. J. Biol. Chem. 263, 9381-9387. 11. Ethier, C.R., Kamm, R.D., Johnson, M., Pavao, A.F., and Anderson, P.J. (1989) Further studies on the flow of aqueous humor through microporous filters. Invest. Ophthalmol. Vis. Sci. 3 J 739-746. L 12. Barsotti, M.F., Bartels, S.P., Freddo, T.F., and Kamm, R.D. (1992) The source of protein in the aqueous humor of the normal monkey eye. Invest. Ophthalmol. Vis. Sci. 3 581-595.
Biologics; and Dr. Alfred0 Aguirre, State University of New York at Buffalo, for the antibody to cystatin.
1243
SHORT COMMUNICATION Volume I 1 number 12 1992, 1239-1243
Protein analysis of monkey aqueous humor
Paul Russell and David L.Epstein'
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
National Eye Institute, National Institutes of Health, Bethesda, MD and 'Department of Ophthalmology, Duke University, Durham, NC, USA
ABSTRACT Aqueous humor from individual eyes of young monkeys (Macaca mulatta) was analyzed by gel exclusion chromatography, and one- and two-dimensional gel electrophoresis. The data indicated that in young normal monkey the main peak of protein eluting from gel exclusion columns was observed around 80,000 daltons. There was a small amount of heavy molecular weight material eluting from these columns with an apparent molecular size of greater than 500,000 daltons, but very little material smaller than 40,000 daltons. By one- and two-dimensional gel electrophoresis, the major polypeptide components in the monkey aqueous were similar to those reported for the human aqueous with polypeptides at around 170, 130, 110, 80, 67, 60, 42, 34, 28, 25, 22, 16, and 14 kilodaltons (kD). The monkey aqueous humor also contains the protease inhibitor cystatin.
guidelines on the use of animals in research. Because of the constraints of the vaccine testing, the animals were first exsanguinated, and immediately after death, a paracentesis of the aqueous humor was done. The paracenteses were done between 8 and 10 AM. A 25 gauge needle on a tuberculin syringe was used with the entry site just anterior to the limbus. The upward-facing bevel of the needle was observed during the removal of the aqueous. Aqueous humor (approximately 100p1) was obtained from each eye, and the individual samples were frozen at -7OOC. Seventy-six samples were used in this study. The protein content of each of the aqueous humor samples was determined using the Bradford method (7).
The aqueous humor of the vertebrate eye plays an essential role in the homeostasis of the tissues in the anterior part of the eye. The composition of the aqueous may play a role in the pathogenesis of cataract or glaucoma and also in wound healing (1-6). Surprisingly little information is available analyzing aqueous humor by gel
Bovine serum albumin was used as a standard. Four of the aqueous samples with a minimum of three aliquots from each were used exclusively for protein measurement. The mean of these four samples was 6.53 mg/dl f 1.77. The other 68 samples were measured using one aliquot of
indication of the apparent native sizes of the proteins in
either 10 or 20 p1. The mean of the 68 other samples was 6.18 mg/dl f 2.38. These values are closer to those reported for human (4) and for other non-human primates
solution. An earlier report on the composition of the
(6) than the value for Rhesus previously reported (3). One
aqueous humor of the Rhesus monkey (Macaca mulatta)
possible reason for the discrepancy in the values may be
suggested that several constituents were very similar to
the use of the different techniques for protein
those reported in human aqueous (3). We have recently
determination. Just as in human, the ascorbate in the
had the opportunity to obtain a number of Rhesus monkey
monkey aqueous humor can interfere with some protein
aqueous specimens, and have extended previous
quantitative methods. For a standard sample of lOmg/dl
observations to individual aqueous samples.
in lmh4 ascorbate, a Lowry protein determination (8) gave
exclusion chromatography. This analysis gives an
Monkeys (Macaca mulatta), aged two to three years,
an inflated protein content of 30.6mg/dl. No interference
were used by the vaccine testing service of the Bureau of
of ascorbate was noted with the Bradford method (data not
Biologics. The monkeys were normal animals. The tests
shown).
were performed in accordance with NIH institutional
High performance liquid chromatography (HPLC)
Received on August 10, 1992; accepted on November 9, 1992
0 Oxford University Press
1239
Current Eye Research kD
-200 -97
-68
-20 -14 0
1 1
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
wR
I\
0 Z
Size (kD)
I 135
F 1 2 3 4 5 6 7 8 91011
1
I
I
67
43
20
Figure 1. The elution pattern of a single sample of monkey aqueous humor eluted from a Superdex 75 column. The polypeptides, eluted from the column, were precipitated in trichloroacetic acid and run on SDS PAGE. The silver
stained gel pattern of the polypeptides in these fractions is shown in the inset. The calibration of the column using protein standards is shown below the figure.
was run using a Superdex 75 column or a Superose 12
little material eluted after 40kD. Polypeptides in the
column (Pharmacia-LKB,Piscataway, NJ) with a flow rate
fractions eluted from the column were precipitated in 10%
of 0.25 ml/min. A variable wave length monitor (LKB
trichloroacetic acid (TCA) by adding a tenth volume of
2151, Pharmacia-LKB) was set at 280nm with a full scale
100%TCA to each fraction. The tubes were cooled to 4OC for a minimum of 30 minutes and then centrifuged at
absorbance of 0.005. Fractions of 500~1were collected. Four different HPLC buffer systems were used. Two
14,000 xg for 20 minutes. The protein pellets were
buffer systems used l O O m M NaCl with either 50mM Tris-
solubilized in 5% sodium dodecylsulfate (SDS) in 62.5mM
HC1 (pH 7.1) or 50mM HEPES (pH 7.1). Both of these buffer systems gave identical results. The other two buffer
Tris-HC1 (pH 8.8) with 5% 0-mercaptoethanol. Typically, each fraction was solubilized in 2p1 of the SDS buffer. The
systems utilized 50 mM Tris-HC1, 100 mM KC1, 1 mh4
sample were analyzed with one-dimensional
EDTA, and 10 mM P-mercaptoethanol at pH values of either 7.1 or 7.4. These two buffer systems gave identical
polyacrylamide gel electrophoresis (PAGE) using the
results to the ones without a reducing agent or EDTA.
were stained with silver as previously reported with the
Superdex 75 chromatography on a 3Opl sample from one eye with a protein content of 5.2 mg/dl revealed a
exception that 5% citric acid was substituted for the 10%
small peak of absorbing material with a relatively large
only unresolved streaks of stain in the first fractions off the
size above about 200kD (Figure 1). There was a separation
column, but some of the heavier molecular weight
of two main absorbing peaks around 130 and 80kD. Very
polypeptides could be resolved beginning with the material
1240
PhastSystem (Pharmacia-LKB). The 12.5%resolving gels
acetic acid in the development solutions (9). There were
Current Eye Research BASIC
ACIDIC
li
kD 9743 -
20-
I A\ FRACT
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
SAMPLE A
E
A 1
1
1
1
1
1
1
1
5
FRACTION 1 I
Size (kD)
11 12 13 14 15 1617 11121314151617 SAMPLE B
1
1
1
1
1
1
I
2000 230
1
1
1
15
10
1
1
20
I
I
I
158
67
25
Figure 2. Su erose 12 column chromatographs of 501-11of a single s a m p i of monkey aqueous with a protein content of 5.8 mg/dl (A) and the two-dimensional gel electrophoresis pattern of the sample (left inset). The aqueous from the fellow eye (4.7mg/dl) was evaporated to dryness,
rehydrated, and then loaded onto the same column (B). Polypeptides precipitated from the fractions of sample B were run on SDS PAGE and are shown by the silver stained gel (right inset). The calibration of the column using protein standards is shown below the figure.
in fraction 4. Faint polypeptide bands around 170, 130 and
aqueous from the fellow eye (B)(4.7mg/dl) that was first
llOkD could be detected. There was a band at 67kD in
evaporated to dryness in a Speed Vac Concentrator
fraction 4 which may have been disulfide linked albumin.
(Savant, Farmingdale, NY) and then solubilized in H,O.
Fraction 5 and 6 showed the main band at 67kD and other
Drying was used to determine if this would adversely
bands around 80 and 60kD; additional polypeptides with
affect the sample. Concentration of the specimens was
smaller molecular weights were present in this fraction.
essential in order to run the two-dimensional
Two or three bands around 35kD as well as bands at 28,
electrophoresis. The chromatogram revealed a small
25,22,16, and 14kdD were observed. Later fractions
amount (4% of the total absorbance) of material eluting in
eluting from the column did not contain very much
the heavy molecular size range at approximately 500kD.
staining material on the SDS gel. Analyses of other
The chromatograph of the rehydrated sample B showed a
individual samples or even pooled samples on this column
somewhat larger amount of material eluting in this range.
gave similar results (n=8).
SDS gel analysis of these fractions revealed only an
To resolve the proteins with larger sizes, multiple
undiscernible smear of stain from the top of the gel down
samples (n=24) were also analyzed with the Superose 12
to about 67kD. The increased amount of material in the
column. The pattern of a sample (A)(5.8mg/dl) run on this
fractions near the start of elution in the samples that had
column (Figure 2) can be compared to the pattern of the
been dehydrated was always small but somewhat variable.
1241
Current Eye Research
I\
kD
E .02
68
-
rn'
14
-
h
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
5 .O I
1L
+ "
I
m
a
S
Figure 3. Elution pattern of pooled monkey aqueous humor from a Superose 12 column and dot blot (below) showing the immunoreactive cystatin eluted from the column primarily in fractions 14 with a weak positive reaction observed in fraction 15. (S) human saliva; positive control. (B) bovine serum albumin; negative control. Dot blots
were done on nitrocellulose. The seam after fraction 13 is the result of joining sequential strips of the dot blot together. (Inset) Silver stained SDS PAGE (I) and western blot (11) of an aqueous sample incubated with antibodies to cystatin. The positions of the molecular weight markers are shown.
Use of elution buffers which contained P-mercaptoethanol had no apparent effect on the elution profiles (data not
similar apparent molecular weights but various isoelectric
shown). The increase of material at this size range may
Several of the polypeptides below 60kD appear to have
represent aggregated protein that could not be sufficiently
very acidic isoelectric points and can be seen near the edge
dissociated during rehydration. However, concentration of
of the gel. Thus, both gel exclusion chromatography and
individual specimens by calibrated filter units such as the
two-dimensional analysis can be done with a single
Centricon-10 units (Amicon, Lexington, MA) resulted in a
aqueous specimen.
significant loss of material from individual samples. There
points may be modified proteins, perhaps glycoproteins.
Cystatin, a 14kD inhibitor of thiol protease, is found
was insufficient material recovered after concentration with
in relatively high quantities in tears and saliva (10).
the Centricon filters to enable further study.
Western blots were done using the protocol of Western
For two-dimensional analysis, an aliquot of sample
Lights (Tropix, Waltham, MA) with the primary antibody
A was concentrated by speedvac and rehydrated in one
diluted 1500. Five individual samples were run on SDS
tenth volume of 8M urea and 2% nonidet P40. The
PAGE and cystatin was detected in all specimens (Figure
sample was applied to the urea isoelectric focusing gel (pH
3). To determine where this small protein was eluting
3-9) and subsequently, the resolved proteins were run on a
from the Superose 12 column, a pooled aliquot of six
12.5% SDS gel (9). The two-dimensional analysis showed
the large proportion of the staining material in the monkey
aqueous samples was used. Dot blots of loop1 of each fraction were incubated with the antiserum for cystatin.
aqueous that was present at 67kD (inset, Figure 2). Several
Immunoreactivity was detected in fractions 14 and 15 in
individual polypeptides can be seen as well as "trains" of
the main peak of absorbance.
polypeptides. These groupings of staining material with
1242
A most interesting finding is the minimal amount of
Current Eye Research lower molecular weight material that i s apparently free in
Supported by grant NIH-NEI RO1-01894 and a grant from
the aqueous. It could be that the sizes of the aggregates
National Glaucoma Research, a Division of the American
that are filtered through the trabecular meshwork are
Health Assistance Foundation, Rockville, Maryland to
tightly controlled. Small hydrophobic proteins could
D.L.E.
potentially interact with the constituents of the trabecular meshwork and lead to the obstruction in flow of the
CORRESPONDING AUTHOR
aqueous (11). The current results would indicate that the
Paul Russell, Bldg. 6 Rm. 228, National Institutes of
bulk of the protein material that must pass through the
Health, Bethesda, MD 20892
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
trabecular meshwork has an apparent native size range between 200 and 40kD. A recent report (12) suggested that the plasma proteins diffuse into the anterior chamber through the anterior iris. If this model is correct, the data presented here indicate a possible role for albumin. Polypeptides having low molecular weights eluted from columns with the albumin fraction. These polypeptides could be aggregating, or they could be associating with albumin. The latter possibility is a very intriguing one since it would suggest that albumin may play an essential role in clearing these components through the trabecular meshwork. Protein-protein interactions within the trabecular meshwork would be lessened if these small proteins were first associated with albumin. This hypothesis would be consistent with the diffusion of the plasma in an area just prior to the outflow pathway. Furthermore, it suggests a previously unappreciated and essential role for albumin in the maintenance of the trabecular meshwork. The data show that the protein content of the Rhesus aqueous humor is nearer to the human value than the higher value previously reported. The results suggest that there are two major protein peaks that elute from gel filtration columns of monkey aqueous. These peaks have sizes of approximately 130 and 80kD and there is little protein material eluting in the size range below 40kD. The polypeptide composition of the Rhesus monkey aqueous appears to be quite similar to the human. ACKNOWLEDGEMENTS We wish to thank Mr. John Cogan of the Bureau of
REFERENCES 1. De Berardinis, E., Tieri, O., Polzella, A., and Iuglio, N. (1965) The chemical composition of the human aqueous humour in normal and pathological conditions. Exp. Eye Res. & 179-186. 2. Sandberg, H.O. and Closs, 0. (1979) The alpha and gamma crystallin content in aqueous humor of eyes with clear lenses and with cataracts. Exp. Eye Res.-82 601-610. 3. Gaasterland, D.E., Pederson, J.E., MacLellan, H.M., and Reddy, V.N. (1979) Rhesus monkey aqueous humor composition and a primate ocular perfusate. Invest. Ophthalmol. Vis. Sci. -8l 1139-1150. 4. Tripathi, R.C., Millard, C.B., and Tripathi, B.J. (1989) Protein composition of human aqueous humor: SDS PAGE analysis of surgical and post-mortem samples. Exp. Eye Res. 48, 117-130. 5. Jampel, H.D., Roche, N., Stark, W.J., and Roberts, A.B. (1990) Transformin growth factor-p in human aqueous humor. Curr. Eye 8es. 3 963-969. 6. Jampel, H.D., Brown, A., Roberts, A., Koya, P., and Quigley, H. (1992) Effect of paracentesis upon the blood-aqueous barrier of cynomolgus monkeys. Invest. Ophthalmol. Vis. Sci. -3 165-171. 7. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principles of protein-dye binding. Anal. Biochem. -27 248-254. 8. Peterson, G.L. (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. € 3 - 346-356. 9. Russell, P. and Yamada, T. (1990) Increased sensitivity of two-dimensional gel electrophoresis using PhastSystem. BioTechniques, 5 422-424. 10. Al-Hashimi, I., Dickinson, D.F, and Levine, M.J. (1988) Purification, molecular cloning, and sequencing of salivary cystatin SA-I. J. Biol. Chem. 263, 9381-9387. 11. Ethier, C.R., Kamm, R.D., Johnson, M., Pavao, A.F., and Anderson, P.J. (1989) Further studies on the flow of aqueous humor through microporous filters. Invest. Ophthalmol. Vis. Sci. 3 J 739-746. L 12. Barsotti, M.F., Bartels, S.P., Freddo, T.F., and Kamm, R.D. (1992) The source of protein in the aqueous humor of the normal monkey eye. Invest. Ophthalmol. Vis. Sci. 3 581-595.
Biologics; and Dr. Alfred0 Aguirre, State University of New York at Buffalo, for the antibody to cystatin.
1243
