Volume 6, number 2
MOLECULAR & CELLULAR BIOCHEMISTRY
February 28, 1975
WATER VAPOR SORPTION OF KERATAN SULFATE Boake PLESSY* and Frederick A. B E T T E L H E I M
Chemistry Department, Adelphi University Garden City, N.Y. 11530 (Received July 9, 1974)
Summary Keratan sulfate was isolated from bovine cornea. The water vapor sorption isotherms were obtained on both sodium and calcium salts of keratan sulfate at different temperatures. Deuterated water sorption isotherms were obtained on sodium keratan sulfates. The infrared spectra of the keratan-sulfate was monitored as a function of water and D 2 0 uptake. The results are discussed in terms of an open polymer matrix that exists in the solid state of keratan sulfate.
the addition of cetylpyridinium chloride 3, which precipitates the glycosaminoglycans, markedly reduces the swelling of cornea. The water sorptive and retentive capacity of different chondroitin sulfates were determined some years ago in this laboratory 4'5. In order to obtain a complete picture on the individual role of the various glycosaminoglycan components it was decided to study the hydration properties of keratan sulfate.
Materials and Methods
Preparation of keratan sulfate Introduction Keratan sulfate is one of the sulfated glycosaminoglycans that occur in connective tissues. It piays a dominating role in the cornea of eyes where it contributes two thirds of the total glycosaminoglycans 1. Although the combined content of keratan sulfate and chondroitin sulfates is only 1% of the fresh cornea, while it contains 78 % water and 15 % collagen, there is ample experimental evidence that this 1% of ground substance is largely responsible for the water binding power of the whole cornea. When the glycosaminoglycans are extracted from the cornea, its swelling and water retention power is much reduced 2. Digestion by hyaluronidase or * Based in part on a thesis submitted to the Graduate School of Adelphi University in partial fulfillment of the requirements for the Ph.D. degree by B. PLESSYwhose permanent address is: Chemistry Department, Dillard University, New Orleans, La., 70122.
Bovine cornea were excised from eyes obtained from slaughterhouses less than 24 hours post mortem. The cornea were stored in acetone. 57 g of the dehydrated cornea were cut into small pieces and reswollen in 500 ml of 0.1 M phosphate buffer at pH 6.5 at room temperature. EDTA and cysteine HC1 were added to make 0.0005 M with respect of each. 13 mg of papain was added per each g of cornea and the slurry was digested at 67°C for 6 hours. The residue was separated from the clear supernatant by centrifugation and discarded. A column of 45 cm long and 4 cm diameter was gravity packed with an aqueous suspension of E C T E O L A . The E C T E O L A , obtained from Biorad Corporation, was purified by washing with 1 N N a O H , 1 N HCI, ethanol and ether in sequence and it was dried before making the aqueous suspension. The supernatant containing the corneal extract was applied directly to this column and washed
Dr. W. Junk b.v. Publishers- The Hague, The Netherlands
85
in with 250 ml distilled water. The glycosaminoglycans were eluted with 0.2 ~ HC1, 2 M ammonium formate and 2 M NaC1 in sequence 6. The appearance of the glycosaminoglycans were monitored by anthrone 7 and carbaz01e 8 test and the protein by ultraviolet absorption at 280 nm. The 2 M NaC1 eluted fraction which contained only keratan sulfate, was precipitated by adding 50 ml 15 ~o sodium acetate and making the total solution 85 ~ with respect to ethanol. After standing overnight at room temperature, the precipitate was harvested by centrifugation and washed with absolute ethanol and subsequently with ether. The preparation was then air dried. A total of 600 mg material was thus obtained. 300 mg of this material was dissolved in 60 ml of 0.5 N sodium acetate and reprecipitated in 85 ~o ethanol. After overnight standing, the precipitate was collected by centrifugation, washed with absolute ethanol and ether and air dried. The harvested material was labelled as sodium salt of keratan sulfate. Another 300 mg of the raw material was dissolved in 60 ml of 2.5 M calcium chloride. The keratan sulfate was precipitated by making the solution 70 ~o with respect to ethanol. After standing overnight at room temperature, the precipitate was collected by centrifugation, washed with absolute ethanol and ether and air dried. This preparation was labelled calcium salt 3f keratan sulfate.
Table I Chemical and physical parameters of corneal keratan sulfate. Hexose % Hexosamine ~ Hexose/hexosamine molar ratio Sulfate/hexosamine molar ratio Weight average molecular weight
23.0 22.0 1.04 1.09 7.300 0.63 cc/mole 51 ml/g
[r/]
obtained at two temperatures about 10°C apart while desorption isotherms were obtained only at the higher temperature. Keratan sulfate films were obtained by casting solutions on a Nichrome plate and allowing a slow evaporation to take place. The film's thickness was measured by micrometer. The film thickness of 0.03 mm did not show any variation between 0.3-0.6 relative vapor pressure. No thickness measurements were attempted at 0 relative vapor pressure. The keratan sulfate film was placed in a vacuum sorption chamber, described earlier 5, that had AgC1 windows for infra red spectroscopy. The temperature as well as the relative humidity
Analysis Molecular weight was determined by sedimentation equilibrium centrifugation in a Beckman Model E analytical centrifuge using ultraviolet absorption optics 9. Hexose content was determined by anthrone 7, total hexosamine content was obtained by a modified Elson-Morgan test 1°. The absence of uronic acid was ascertained by the negative result of the" carbazole test 8. Sulfate was obtained through a turbidimetric method 1°. Intrinsic viscosity was obtained from the extrapolation of the nsp/C vs c curve. The viscosity of the solutions in 0.1 N NaC1 was measured in a CannonUbbelhode capillary viscometer, (Table I).
//
L
o
Water vapor sorption The H 2 0 and D 2 0 vapor sorption isotherms were obtained gravimetrically in an apparatus described earlier 4. Sorption isotherms were 86
I
__
o!2
½
'
0!~
J
o'0
Fig. 1. Water vapor sorption isotherms of calcium salts of keratan sulfate in terms of g H20 per 100 grams of keratan sulfate vs relative vapor pressure. ~ sorption at 29.6°C ©--© desorption at 29.6°C; 0~---(~sorption at 16.1°C.
of the chamber was controlled and monitored throughout the experiment. The two cells of the vacuum chamber fitted nicely in the Beckmann IR-7 spectrophotometer and the spectra were recorded in double deam set-up with NaC1 optics. The films were suspended in the sample arm of the vacuum cell and were dried by evacuating the system. The integrated band intensity of the O---H stretching mode at 3450 cm-1 was monitored throughout the drying process and reproducibility of this band during 24 hours drying period was used as the criteria for dryness. Drying was complete in 72-90 hours. After the evacuation process, the spectrum of the film was recorded at zero relative vapor pressure. Water vapor was introduced into the system and the system was allowed to equilibrate. After equilibrium was established but in no case less than 24 hours, another spectrum was recorded. Approximately six spectra were obtained from zero to 0.6 relative vapor pressure, Desorption isotherms were then obtained on the same films after which the sorption branch of the isotherms were duplicated. Thus, the reproducibility of the sorption isotherm was clearly established. / 3C
,
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20 ///
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10
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I
0.2
I
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I
0,4
I
0,6
Fig. 2. Water vapor sorption isotherms of sodium salt keratan sulfate: g of water sorbed per 100 grams of keratan sulfate vs. relative vapor pressure 0---0 sorption and 26.6°C; O---
MOLECULAR & CELLULAR BIOCHEMISTRY
February 28, 1975
WATER VAPOR SORPTION OF KERATAN SULFATE Boake PLESSY* and Frederick A. B E T T E L H E I M
Chemistry Department, Adelphi University Garden City, N.Y. 11530 (Received July 9, 1974)
Summary Keratan sulfate was isolated from bovine cornea. The water vapor sorption isotherms were obtained on both sodium and calcium salts of keratan sulfate at different temperatures. Deuterated water sorption isotherms were obtained on sodium keratan sulfates. The infrared spectra of the keratan-sulfate was monitored as a function of water and D 2 0 uptake. The results are discussed in terms of an open polymer matrix that exists in the solid state of keratan sulfate.
the addition of cetylpyridinium chloride 3, which precipitates the glycosaminoglycans, markedly reduces the swelling of cornea. The water sorptive and retentive capacity of different chondroitin sulfates were determined some years ago in this laboratory 4'5. In order to obtain a complete picture on the individual role of the various glycosaminoglycan components it was decided to study the hydration properties of keratan sulfate.
Materials and Methods
Preparation of keratan sulfate Introduction Keratan sulfate is one of the sulfated glycosaminoglycans that occur in connective tissues. It piays a dominating role in the cornea of eyes where it contributes two thirds of the total glycosaminoglycans 1. Although the combined content of keratan sulfate and chondroitin sulfates is only 1% of the fresh cornea, while it contains 78 % water and 15 % collagen, there is ample experimental evidence that this 1% of ground substance is largely responsible for the water binding power of the whole cornea. When the glycosaminoglycans are extracted from the cornea, its swelling and water retention power is much reduced 2. Digestion by hyaluronidase or * Based in part on a thesis submitted to the Graduate School of Adelphi University in partial fulfillment of the requirements for the Ph.D. degree by B. PLESSYwhose permanent address is: Chemistry Department, Dillard University, New Orleans, La., 70122.
Bovine cornea were excised from eyes obtained from slaughterhouses less than 24 hours post mortem. The cornea were stored in acetone. 57 g of the dehydrated cornea were cut into small pieces and reswollen in 500 ml of 0.1 M phosphate buffer at pH 6.5 at room temperature. EDTA and cysteine HC1 were added to make 0.0005 M with respect of each. 13 mg of papain was added per each g of cornea and the slurry was digested at 67°C for 6 hours. The residue was separated from the clear supernatant by centrifugation and discarded. A column of 45 cm long and 4 cm diameter was gravity packed with an aqueous suspension of E C T E O L A . The E C T E O L A , obtained from Biorad Corporation, was purified by washing with 1 N N a O H , 1 N HCI, ethanol and ether in sequence and it was dried before making the aqueous suspension. The supernatant containing the corneal extract was applied directly to this column and washed
Dr. W. Junk b.v. Publishers- The Hague, The Netherlands
85
in with 250 ml distilled water. The glycosaminoglycans were eluted with 0.2 ~ HC1, 2 M ammonium formate and 2 M NaC1 in sequence 6. The appearance of the glycosaminoglycans were monitored by anthrone 7 and carbaz01e 8 test and the protein by ultraviolet absorption at 280 nm. The 2 M NaC1 eluted fraction which contained only keratan sulfate, was precipitated by adding 50 ml 15 ~o sodium acetate and making the total solution 85 ~ with respect to ethanol. After standing overnight at room temperature, the precipitate was harvested by centrifugation and washed with absolute ethanol and subsequently with ether. The preparation was then air dried. A total of 600 mg material was thus obtained. 300 mg of this material was dissolved in 60 ml of 0.5 N sodium acetate and reprecipitated in 85 ~o ethanol. After overnight standing, the precipitate was collected by centrifugation, washed with absolute ethanol and ether and air dried. The harvested material was labelled as sodium salt of keratan sulfate. Another 300 mg of the raw material was dissolved in 60 ml of 2.5 M calcium chloride. The keratan sulfate was precipitated by making the solution 70 ~o with respect to ethanol. After standing overnight at room temperature, the precipitate was collected by centrifugation, washed with absolute ethanol and ether and air dried. This preparation was labelled calcium salt 3f keratan sulfate.
Table I Chemical and physical parameters of corneal keratan sulfate. Hexose % Hexosamine ~ Hexose/hexosamine molar ratio Sulfate/hexosamine molar ratio Weight average molecular weight
23.0 22.0 1.04 1.09 7.300 0.63 cc/mole 51 ml/g
[r/]
obtained at two temperatures about 10°C apart while desorption isotherms were obtained only at the higher temperature. Keratan sulfate films were obtained by casting solutions on a Nichrome plate and allowing a slow evaporation to take place. The film's thickness was measured by micrometer. The film thickness of 0.03 mm did not show any variation between 0.3-0.6 relative vapor pressure. No thickness measurements were attempted at 0 relative vapor pressure. The keratan sulfate film was placed in a vacuum sorption chamber, described earlier 5, that had AgC1 windows for infra red spectroscopy. The temperature as well as the relative humidity
Analysis Molecular weight was determined by sedimentation equilibrium centrifugation in a Beckman Model E analytical centrifuge using ultraviolet absorption optics 9. Hexose content was determined by anthrone 7, total hexosamine content was obtained by a modified Elson-Morgan test 1°. The absence of uronic acid was ascertained by the negative result of the" carbazole test 8. Sulfate was obtained through a turbidimetric method 1°. Intrinsic viscosity was obtained from the extrapolation of the nsp/C vs c curve. The viscosity of the solutions in 0.1 N NaC1 was measured in a CannonUbbelhode capillary viscometer, (Table I).
//
L
o
Water vapor sorption The H 2 0 and D 2 0 vapor sorption isotherms were obtained gravimetrically in an apparatus described earlier 4. Sorption isotherms were 86
I
__
o!2
½
'
0!~
J
o'0
Fig. 1. Water vapor sorption isotherms of calcium salts of keratan sulfate in terms of g H20 per 100 grams of keratan sulfate vs relative vapor pressure. ~ sorption at 29.6°C ©--© desorption at 29.6°C; 0~---(~sorption at 16.1°C.
of the chamber was controlled and monitored throughout the experiment. The two cells of the vacuum chamber fitted nicely in the Beckmann IR-7 spectrophotometer and the spectra were recorded in double deam set-up with NaC1 optics. The films were suspended in the sample arm of the vacuum cell and were dried by evacuating the system. The integrated band intensity of the O---H stretching mode at 3450 cm-1 was monitored throughout the drying process and reproducibility of this band during 24 hours drying period was used as the criteria for dryness. Drying was complete in 72-90 hours. After the evacuation process, the spectrum of the film was recorded at zero relative vapor pressure. Water vapor was introduced into the system and the system was allowed to equilibrate. After equilibrium was established but in no case less than 24 hours, another spectrum was recorded. Approximately six spectra were obtained from zero to 0.6 relative vapor pressure, Desorption isotherms were then obtained on the same films after which the sorption branch of the isotherms were duplicated. Thus, the reproducibility of the sorption isotherm was clearly established. / 3C
,
///
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/
//
iii/I
/1/~11
20 ///
%
/7 Z////O//
10
///o
I
I
0.2
I
~/~o
I
0,4
I
0,6
Fig. 2. Water vapor sorption isotherms of sodium salt keratan sulfate: g of water sorbed per 100 grams of keratan sulfate vs. relative vapor pressure 0---0 sorption and 26.6°C; O---
