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Biochimica et Biophysica Acta, 543 (1978) 258--263
© Elsevier/North-Holland Biomedical Press
BBA 28625 WATER BINDING BY G L Y C O G E N MOLECULES
T. BRITTAIN and R. GEDDES Department of Biochemistry, University of Auckland, Auckland (New Zealand)
(Received January 26th, 1978)
Summary L o w temperature NMR spectra have been obtained of the water b o u n d to glycogen. These data have allowed the evaluation of the amount of water b o u n d and the energy and entropy associated with this bonding. High molecular weight glycogen (approx. 1 • 109) exhibits water binding properties analogous to those previously found for other glycoproteins. Low molecular weight glycogen (approx. 1 • 10~), however, shows anomalous binding characteristics, with large amounts of associated "non-freezing" water. These findings are disCussed in terms of previously proposed molecular architecture. Introduction The a m o u n t of water b o u n d b y certain proteins and a glycoprotein has recently been measured b y NMR [1]. It was implied that the hydration of the Carbohydrate portion of the glycoprotein was 0.66 g per g carbohydrate, which is relatively close to the 0.59 g per g carbohydrate previously reported for Agarose [2]. Additionally, a recent h y d r o d y n a m i c study of liver glycogen [3] has reported 0.3--1.1 g water associated with each g polysaccharide. Since this latter study measured water b o u n d under specific hydrodynamic conditions (capillary viscometry, zonal centrifugation, etc.), and since this t y p e of b o u n d water is some sort of average, it could well be different under varied hydrodynamic conditions [4]. Therefore it seemed of interest to investigate the hydration of glycogen b y NMR. Further, it proved impossible to estimate the a m o u n t of water b o u n d to low molecular weight glycogen b y hydrodynamic means, and as this is probably the most c o m m o n molecular size in the cytosol [5] it was also of great interest to ascertain this quantity. Experimental Livers were quickly removed from rabbits (New Zealand White), which had been given an overdose of Nembutal ( A b b o t t Laboratories Ltd, Naenae, New
259 Zealand) and plunged into liquid nitrogen. Glycogen was then isolated from the tissue by cold-water extraction [5,6] and fractionated on a sucrose density gradient [3,5]. Fractions were combined to provide both " l o w " and " h i g h " molecular weight samples. Approximate molecular weights were calculated as detailed in reference [3]. Glycogen concentrations were determined either by an iodine-iodide reaction (in the presence of sucrose) [6] or by a standard anthrone m e t h o d (after extensive dialysis of the glycogen samples). All samples were made 10 mM in KC1 prior to the recording of NMR spectra. The samples were placed into 10 mm commercial NMR tubes and a sample of deutero-acetone in a coaxial 3-mm tube acted as an external reference lock. Spectra were recorded using a JEOL FX60 60 MHz spectrometer. In order to obtain Fourier transform spectra the samples were pulsed 30 times using a 10 kHz spectral width. The samples were maintained to within +0.5°C of the required temperature by use of the JEOL NM5471 temperature programmer. At each stage of cooling, 20 min were allowed for equilibration of the sample after the temperature probe had attained the required reading. The line widths reported here represent the width (in Hz) at half height of the spectral curves. Areas were calculated as the product of the line width and height and were converted to hydration values by comparison with a standard water sample, in this case bovine serum albumin. The value of 0.37 g water/g protein a t - - 3 5 ° C was used for bovine serum albumin [ 1 ].
Results and Discussion Fig. 1 shows clearly t h a t even though the bulk water in the samples used has frozen at --15°C, a measurable NMR water signal is still apparent in glycogen. This is consistent with previous data on a variety of macromolecules which have been shown to possess a certain a m o u n t of " b o u n d " water which does n o t freeze at temperatures down to --35°C [1,7--9]. However, differences are apparent in the non-freezing water in the samples. Fractions C and A of glycogen (Mr of approx. 1 . 1 0 7 and 1 . 1 0 9 , respectively [3]). The smaller line width obtained for fraction A shows a greater mobility of the associated water compared to fraction C. The line widths of the various low molecular weight fractions of glycogen show a linear dependence on temperature in Fig. 2. From these Arrhenius plots it is apparent that whole glycogen and fraction C have activation energies similar to bovine serum albumin. Fig. 3 shows that the a m o u n t of water associated with these fractions is very similar to t h a t of bovine serum albumin [1], being approx. 0.35 g water/g glycogen at --35 ° C. Molecular weight fractions B (Mr approx. 5 • 107 [3]) and A, however, show NMR signals different from bovine serum albumin {Fig. 1). Fig. 2 shows t h a t the line widths for these fractions are much smaller than those of bovine serum albumin at the same temperature, and are almost temperature independent. Analysis of the areas of the NMR curves for fractions B and A show a level of h y d r a t i o n of approximately 0.8 g water/g glycogen and 9 g water/g glycogen, respectively, at --35 ° C. These data suggest t h a t in the sample of whole glycogen the contributions arising from fraction A in particular must be very small. This
260
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~35o ~L
Biochimica et Biophysica Acta, 543 (1978) 258--263
© Elsevier/North-Holland Biomedical Press
BBA 28625 WATER BINDING BY G L Y C O G E N MOLECULES
T. BRITTAIN and R. GEDDES Department of Biochemistry, University of Auckland, Auckland (New Zealand)
(Received January 26th, 1978)
Summary L o w temperature NMR spectra have been obtained of the water b o u n d to glycogen. These data have allowed the evaluation of the amount of water b o u n d and the energy and entropy associated with this bonding. High molecular weight glycogen (approx. 1 • 109) exhibits water binding properties analogous to those previously found for other glycoproteins. Low molecular weight glycogen (approx. 1 • 10~), however, shows anomalous binding characteristics, with large amounts of associated "non-freezing" water. These findings are disCussed in terms of previously proposed molecular architecture. Introduction The a m o u n t of water b o u n d b y certain proteins and a glycoprotein has recently been measured b y NMR [1]. It was implied that the hydration of the Carbohydrate portion of the glycoprotein was 0.66 g per g carbohydrate, which is relatively close to the 0.59 g per g carbohydrate previously reported for Agarose [2]. Additionally, a recent h y d r o d y n a m i c study of liver glycogen [3] has reported 0.3--1.1 g water associated with each g polysaccharide. Since this latter study measured water b o u n d under specific hydrodynamic conditions (capillary viscometry, zonal centrifugation, etc.), and since this t y p e of b o u n d water is some sort of average, it could well be different under varied hydrodynamic conditions [4]. Therefore it seemed of interest to investigate the hydration of glycogen b y NMR. Further, it proved impossible to estimate the a m o u n t of water b o u n d to low molecular weight glycogen b y hydrodynamic means, and as this is probably the most c o m m o n molecular size in the cytosol [5] it was also of great interest to ascertain this quantity. Experimental Livers were quickly removed from rabbits (New Zealand White), which had been given an overdose of Nembutal ( A b b o t t Laboratories Ltd, Naenae, New
259 Zealand) and plunged into liquid nitrogen. Glycogen was then isolated from the tissue by cold-water extraction [5,6] and fractionated on a sucrose density gradient [3,5]. Fractions were combined to provide both " l o w " and " h i g h " molecular weight samples. Approximate molecular weights were calculated as detailed in reference [3]. Glycogen concentrations were determined either by an iodine-iodide reaction (in the presence of sucrose) [6] or by a standard anthrone m e t h o d (after extensive dialysis of the glycogen samples). All samples were made 10 mM in KC1 prior to the recording of NMR spectra. The samples were placed into 10 mm commercial NMR tubes and a sample of deutero-acetone in a coaxial 3-mm tube acted as an external reference lock. Spectra were recorded using a JEOL FX60 60 MHz spectrometer. In order to obtain Fourier transform spectra the samples were pulsed 30 times using a 10 kHz spectral width. The samples were maintained to within +0.5°C of the required temperature by use of the JEOL NM5471 temperature programmer. At each stage of cooling, 20 min were allowed for equilibration of the sample after the temperature probe had attained the required reading. The line widths reported here represent the width (in Hz) at half height of the spectral curves. Areas were calculated as the product of the line width and height and were converted to hydration values by comparison with a standard water sample, in this case bovine serum albumin. The value of 0.37 g water/g protein a t - - 3 5 ° C was used for bovine serum albumin [ 1 ].
Results and Discussion Fig. 1 shows clearly t h a t even though the bulk water in the samples used has frozen at --15°C, a measurable NMR water signal is still apparent in glycogen. This is consistent with previous data on a variety of macromolecules which have been shown to possess a certain a m o u n t of " b o u n d " water which does n o t freeze at temperatures down to --35°C [1,7--9]. However, differences are apparent in the non-freezing water in the samples. Fractions C and A of glycogen (Mr of approx. 1 . 1 0 7 and 1 . 1 0 9 , respectively [3]). The smaller line width obtained for fraction A shows a greater mobility of the associated water compared to fraction C. The line widths of the various low molecular weight fractions of glycogen show a linear dependence on temperature in Fig. 2. From these Arrhenius plots it is apparent that whole glycogen and fraction C have activation energies similar to bovine serum albumin. Fig. 3 shows that the a m o u n t of water associated with these fractions is very similar to t h a t of bovine serum albumin [1], being approx. 0.35 g water/g glycogen at --35 ° C. Molecular weight fractions B (Mr approx. 5 • 107 [3]) and A, however, show NMR signals different from bovine serum albumin {Fig. 1). Fig. 2 shows t h a t the line widths for these fractions are much smaller than those of bovine serum albumin at the same temperature, and are almost temperature independent. Analysis of the areas of the NMR curves for fractions B and A show a level of h y d r a t i o n of approximately 0.8 g water/g glycogen and 9 g water/g glycogen, respectively, at --35 ° C. These data suggest t h a t in the sample of whole glycogen the contributions arising from fraction A in particular must be very small. This
260
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~35o ~L
