CHYOBIOLOGY
12,
276-281 (19%)
BRIEF COMMUNICATION The Results of Freezing and Dehydration of l-lorseradish Peroxidase BEN DARBYSHIRE Scientific and Indu.striaE Research Organization, Division Resend, Private Mail Bag, Grifith, N.S.W. 268Q, Australia
Commonwealth Irrigation
The use of a pressure membrane apparatus to quantitativeIy dehydrate enzymes prompted a comparison of freezing and dehydration effects on catalase (EC 1.11.1.6) (7). Th is investigation supported previous reports (II, 14, 16) that freezing couId result in dehydration of biological systems. This paper reports the results of experiments where the effects of freezing of horseradish peroxidase (EC 1.11.1.7) are compared with dehydration effects. The pyrroprotective influence of polyvinyl lidone (PVP) and the consequences of different protein levels were examined. METHODS
Horseradish peroxidase (EC 1.11.1.7) was purchased from the Sigma ChemicaI Company. The enzyme was taken up in either sodium phosphate buffer (0.1 M, pH 6.4), sodium acetate buffer (0.1 M, pH 5.6 ), or distilled water to give a final protein concentration of 0.4 pg/ml. Enzyme assay. The reaction mixture consisted of 3 ml of a solution containing 200 ~1 of 1% o-dianisidine in 25 ml of 0.03% hydrogen peroxide and the reaction started by the addition of 100 ,uI of enzyme preparation, Activity was recorded as change in absorbance at 460 nm during 1 min using a Perkin-Elmer Mode1 402 spectrophotometer. Received August 14, 1974.
Freezing technique. Enzyme preparations, in either the acetate or phosphate buffer or water and at different protein concentrations or containing PVP at various concentrations, were frozen to -20°C. Freeze-thaw times were 40- 30-min duration, respectively. Thawing was allowed to occur at room temperature. Five freezethaw cycles were completed and the volume of enzyme preparation subjected to this treatment was 400 ,uI. Dehydration technique. While this technique has already been described in detail elsewhere (5, 6, 7, 23), it is desirable to repeat details of the method so that the process can be completely understood. A pressure membrane apparatus (5) has been used to manipulate the water potential of the enzyme preparation. (Water potential, as defined by Slatyer and Taylor (22), refers to the difference between the partial specific Gibbs free energy of water in a system compared with that of pure water. Dehydration to -2 bars is much less severe than dehydration to -10 bars, ) The pressure membrane apparatus consists of a cylindrical brass body closed at one end which contains a solid fiIter-support leading to a drain, A dialysis membrane (26/ 32) underlain by a Millpore filter (0.01 pm) is held in position over the filter support by an acrylic sleeve containing a Teflon insert which defines an effective filter area of 25mm diameter. 276
Copyright All rights
0 1975 by Academic Press, of reproduction in any form
Inc. reserved.
of
FREEZING
AND
DEHYDRATION
In the case of peroxidase, 400 pl of the enzyme preparation were added to the apparatus and a selected hydrostatic pressure applied. This increased the water potential of the system, and water and small solute molecules passed through the membrane. Equilibration was allowed to occur for 90 min when the enzyme system inside the chamber was in equiIibrium with the solution extomal to the chamber and at atmospheric pressure. The hydrostatic pressure was then released subjecting the internal enzyme phase to a tension equivalent to the applied pressure. The enzyme system was then rehydrated with 400 ,.J of the appropriate buffer, Hence, when different hydrostatic pressures were applied, the peroxidase preparation could be dehydrated to a range of water potentials, After rehydration the preparation was assayed and thus the effect of the dehydration treatment determined. Controls for this treatment consisted of subjecting an enzyme preparation to a hydrostatic pressure equivalent to the preparation being dehydrated. As reported previously with cataIase (7)) hydrostatic pressure aIone had no effect on enzyme activity, and all manipulations were conducted at 25°C. Ulfrauiolet difference-spectra. This technique, using matched tandem celIs in a Perkin-Elmer 402 double-beam spectrophotometer, was employed to determine if the freezing or dehydration treatments re-
Cycle number FIG. I. The c:ffect of freeze-thaw cycle number on the inactivation of peroxidase in different buffering systems. O-0, Phosphate; x-x, acetate; a-*, water.
OF PEROXIDASE
277
TABLE
1
PEROXIDME ACTIVITY EXPRKSWII s PEHCENT CONTROLS AFTER V+~RIOUS TREXPMENTG Treatment
OF
Dehydration to -10 bars
Freea-thaw 5 Cycles
33.2
53.0
23.4 12.0
44.0 38.0
Phosphate buffer, pH 6.4, 0.1 M Acetate buffer, pH 5.6, 0.1 Y Water
sulted in conformational protein molecules ( 12).
changes in the
Protein determination. The method of Lowry et al. ( 19) was used for all protein determinations. Polyacrylamide-gel electrophoresis. Discontinuous electrophoresis in 53% polyacrylamide gels was carried out in the Tris-glycine system of Davis (9). A current of 2 mA per tube was used and the peroxidase isozymes stained with benzidene-H302 ( 17). RESULTS
AND
DISCUSSION
It was found initially that five freezethaw cycles reduced the activity of peroxidase to a greater extent than a single cycle (Fig. 1). Dehydration of peroxidase over a range of water potentials (-4 to -15 bars) generated similar activity losses, so dehydration to -10 bars was arbitrariIy chosen to compare freezing and dehydration. A comparison of the effects of freezing and dehydration are presented in Table 1. All protein was recovered foIIowing the dehydration treatment, and it can be seen from Table 1 that the effect of dehydration was more severe than freezing. However it is interesting to observe that in both cases the presence of buffer ions protected the enzyme to some extent from inactivation. As Connor and Ashwood-Smith (4) pointed out, following the results of Lovelock (18) many people attributed the damaging effect of freezing to high salt concentrations. In the ifl vitro experiments reported in this paper
BEN DARBYSHIRE
278
*/a P v F!
FIG. 2. The effect of different concentrations of PVP on the stability of peroxidase following five freeze-thaw cycles. Symbols as for Fig. 1.
buffered enzyme preparations were less affected by dehydration and freezing than preparations in water alone. The protective effects of poIyviny1 pyrroIidone (PVP), at different concentrations, against damage due to freezing and dehydration were determined. Figure 2 indicates that a concentration of PVP of 0.01% was most effective in protecting the enzyme from freezing, At higher concentrations the protection afforded by PVP was maintained when the enzyme preparation was in water, However, in the presence of phosphate or acetate buffers, the effectiveness of PVP in preventing damage decreased. Mackenzie and Rasmussen (20) reported that at Iower temperatures PVP tended to be more hydrated. Perhaps at higher PVP concentrations, PVP and the enzyme molecules are competing for water molecules to maintain hydration,
FIG. 3. The protection of peroxidase against dehydration to -10 bars by increasing concmtrations of PVP. Symbols as for Fig. 1.
FIG. 4. The relationship between increasing protein concentration on the stability of peroxidase after five freeze-thaw cycles. Symbols as for Fig. 1.
and this results in an increased dehydration of peroxidase when frozen. The protective effect of different concentrations of PVP on dehydration of peroxidase is shown in Fig. 3. Protection increases until 1% PVP and higher concentrations did not result in reduced protection. Thus, PVP will protect peroxidase against the effects of both freezing and dehydration, but different concentrations are required to achieve maximum protection. To determine if protein-protein interactions influenced the effects of freezing and dehydration, the concentration of protein was increased from 0.4 pg/ml, as used in the above experiments, to higher values. Figure 4 indicates the effect of increasing protein concentration when peroxidase is frozen. It can be seen that in the presence of acetate or phosphate buffers, when the protein concentration is increased to between 2 and 4 pg/ml, the enzyme is no longer susceptible to freezing damage. In water, a concentration of at least 10 pg/ml was required to remove freezing damage. When peroxidase was dehydrated, increased protein concentration again protected the enzyme but a higher concentration of 20 pg/ml was required to achieve approximately 80% recovery of activity (Fig. 5). Ansevin and Lauffer (1) reported that low temperatures, low ionic concentrations
FREEZING
AND
DEHYDRATION
and IOW protein concentrations resulted in a depoIymerisation of tobacco mosaic virus protein. Later Laufkr (13) reported that when tobacco mosaic virus protein and insulin interacted to polymerize, release or exclusion of water occurred. It is suggested that a similar mechanism operates when increasing protein concentration results in the protection of peroxidase from the effects of dehydration and freezing. At higher concentrations, interaction of protein m0Iecules forming an ohgomeric protein may lead to a greater stability of the peroxidase molecules with less associated water and hence more recoverabIe activity during periods of desiccation. Ultraviolet difference-spectra did not reveal any conformation differences between the native and dehydrated or frozen preparations. It is possible that conformationaI changes following desiccation treatments may not have been detected due to the comparatively low concentrations of protein required before damage occurred, Similarly, polyacrylamide-gel electrophoresis was not a viable method to determine if particular isozymes were more sensitive to desiccation than others. This was because protein concentrations high enough to detect on gels protected the enzyme from either freezing or dehydration, It may require fluorescent probe analysis to determine if conformational changes were the cause of inactivation of peroxidase following desiccation. In addition, to determine if individual isozymes differ in their desiccation susceptibilities, it will most probably be necessary to separate the isozymes before subjecting them to freezing or dehydration. It has been reported (8) that the carbohydrate units of three fungaI enzymes protect these molecules against the adverse effects of dehydration. Although horseradish peroxidase is glycosylated (21) and the carbohydrate portion accounts for 18% by weight of the molecule, in this case no immediate indication of protection by the
a a&
OF PEROXIDASE
/ o:1 OL
279
I I, I I 20 20 ‘I LW Protwn con~cotrot~onl&g /mi )
FIG. 5. The effect
of dehydration
of peroxidase
to -10 bars at increasing concentrations of protein. Symbols as for Fig. 1. sugar moieties against freezing or dehydration is apparent. However carbohydrates may play a role as the protein concentration is increased, either directly by protection or by assisting in a polymerization mechanism. The interpretation of the damage due to freezing and dehydration remains essentially the same as reported previously (7). Water in the presence of macromolecules is structured (3, 10, 15, 24) and plays a part in maintaining the tridimensional structure of proteins (24). Water removal by freezing or dehydration results in an irreversibIe change in the molecule causing enzyme inactivation, In the results persented here higher protein concentrations may stabilize the molecules through protein-protein interactions and water release. The presence of optimal 1eveIs of PVP could reduce the vapour-pressure difference between the protein phase and the external environment ( 16). PVP may also interact directly with protein to form a more stable complex containing a reduced water content. In the case of freezing of peroxidase, where increasing concentrations of PVP reduced its protective effect, PVP may be competing for water molecules and result in further dehydration of the enzyme. Thus the damage caused by both the freezing and dehydration of peroxidase ilz vitro may be interpreted as a dehydration
BEN DARBYSHIRE
280
process. Further work will be required to investigate the contribution of the ionic environment to the protection of the enzyme against desiccation and to expIain fully the protective mechanism of increasing protein concentrations,
sure
membrane
device.
Lab. Pratt.
6. Darbyshire, B., and Steer, B. T. Dehydration of macromolecules, I. Effect of dehydration-rehydration on indoleacetic acid oxidase, ribonuclease, ribulosediphosphate carboxylase and ketose-I-phosphate aldolase.
AIL&. 1. Biol. Sci. 26, 591404 SUMMARY
Explanations of the mechanism of freezing injury have included the one that freezing may result in dehydration of enzymes. This hypothesis has been examined by comparing the effects of freezing and dehydration on horseradish peroxidase. It was found that freezing and dehydration reduce the activity of peroxidase when compared with the native enzyme. Polyvinyl pyrrolidone and increased protein concentration protect the enzyme against loss of activity in both treatments, Proteinprotein interactions exclude water, and this mechanism is suggested to stabilize peroxidase and protect against desiccation. Polyvinyl pyrrolidone may protect against freezing by reducing dehydration through reduced vapor-pressure difference or may stabilize by a protein-polymer interaction. ACKNOWLEDGMENTS I thank Mrs. Lynette technical assistance and protein determinations.
Dewar far competent Mr. Leith Higgins for
REFERENCES 1. Ansevin, A. T., and Lauffer, M. A. Native tobacco mosaic virus protein of molecular weight 18,000. NUttLTe (London) 183, lf301-1602 (1959). 2. Ashwood-Smith, M. J., and Warby, C. Protective effect of low and high molecular weight compounds on the stability of catalase subjected to freezing and thawing. CTyobioEogy 9, 137-140 ( 1972). Q. Chapman, G., and McLauchlan, K. A. Orientated water in the sciatic nerve of rabbit. Nature (London) 215, 391392 (1967). 4. Connor, W., and Ashwood-Smith, M. J. Cryoprotection of mammalian cells in tissue culture with polymers; possibIe mechanisms. CqobioEogy 10, 488-496 ( 1973). 5. Darbyshire, B., and Anlezark, R. N. A simple and versatile pressure fihering and pres-
21,
554 (1972).
(1973).
7. Darbyshire, B. The influence of dehydration on catalase stability. A comparison with freezing effects. Cryobiology II, 148-151 ( 1974). 8. Darbyshire, B. The function of the carbohydrate units of three fungal enzymes in their resistance to dehydration. Plant Yhysiol. 54, 717-721 (1974). 9. Davis, B. J. Disc electrophoresis. II. Method and application to human serum proteins, Ann. N.Y. Acad. Sci. 121, 404427 (1964). 10. Grant, E. H. The structure of water neighboring proteins, peptides and amino acids as deduced from dielectric measurements. Ann. N.Y. Acad. Sci. 125, 418-427 (1965). II. Heber, U. Freezing injury in relation to loss of enzyme activities and protection against freezing. Cryobi0Z0g~ 5, 188-201 (1968). 12. Kronman, M. J., and Robbins, M. Buried and exposed groups in proteins. In “Fine Structure of Proteins and Nucleic Acids” (G. D. Fasman and S. N. Timasheff, Eds.) pp, 271-416. Dekker, New York, 1970. 13. Lauffer, M. A. Protein-protein interaction: Endothermic polymerization and biological processes. In “Proteins and Their Reactions” (H. W. Schultz and A. F. Anglemier, Eds.), pp. 87-116. Avi Publishing Company, Westport, Conn., 1964. 14. Levitt, J. “The Hardiness of Plants.” Academic Press, New York 1956. 15. Ling, G. N. Hydration of macromolecules. In “Water and Aqueous Solutions. Structure, Thermodynamics and Transport Processes” (R. A. Horne, Ed. ), pp, 683-700, WiIey-Interscience, New York, 1972. 16. Litvan, G. G. Mechanism of cryoinjury in biological systems. Cryobiology 9, 182-191. (1972). 17. Liu, E. H. A simple method for determining the relative activities of individual peroxidase isozymes in a tissue extract. A&. Biochem. 56, 149-X4 ( 1973). 18. Lovelock, J. E. The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Biochtm. Biophys. Acta 11, 28-36 (1953). 19. Lowry, 0. H., Rosebrough, N. J., Fan; A. L., and Randall, R. j. Protein measurement
FREEZING
AND
DEHYDRATION
with the FoIin phenol reagent. .I. Biol. Cham. 193, 265-275 (1951). 20. MacKenzie, A. P., and Rasmussen, 13. H. Interactions in the water-poIyviny1 pyrrolidone system at low temperatures. In “Water Structure at the Water-Polymer Interface” (H. H. G. Jelhnek, Ed.) pp. 146-172. PIenum, New York, 1972. 21. Shannon, L. M., Kay, E., and Lew, J. Y. Peroxidase isozymes from horseradish roots. I. Isolation and physical properties. J. Biol.
Chew. 241, 2166-2172 (1966).
OF PEROXIDASE
281
22. Slatyer, R. O., and Taylor, S. A. Terminology in plant and soil-water relations. Nature (London) 187, 922-924 ( 1960). 23. Steer, B. T. Dehydration of Macromolecules. II. The protective effect of certain anions on ribulosediphosphate carboxylase subjected to low water potentials in vitro. AZ&. J. Bid. Sci. 26, 1435-1442 ( 1973). 24. Tait, M. J., and Franks, F. Water ical systems. Nature (Londo~z) (1971).
and biolog230, 91-94
12,
276-281 (19%)
BRIEF COMMUNICATION The Results of Freezing and Dehydration of l-lorseradish Peroxidase BEN DARBYSHIRE Scientific and Indu.striaE Research Organization, Division Resend, Private Mail Bag, Grifith, N.S.W. 268Q, Australia
Commonwealth Irrigation
The use of a pressure membrane apparatus to quantitativeIy dehydrate enzymes prompted a comparison of freezing and dehydration effects on catalase (EC 1.11.1.6) (7). Th is investigation supported previous reports (II, 14, 16) that freezing couId result in dehydration of biological systems. This paper reports the results of experiments where the effects of freezing of horseradish peroxidase (EC 1.11.1.7) are compared with dehydration effects. The pyrroprotective influence of polyvinyl lidone (PVP) and the consequences of different protein levels were examined. METHODS
Horseradish peroxidase (EC 1.11.1.7) was purchased from the Sigma ChemicaI Company. The enzyme was taken up in either sodium phosphate buffer (0.1 M, pH 6.4), sodium acetate buffer (0.1 M, pH 5.6 ), or distilled water to give a final protein concentration of 0.4 pg/ml. Enzyme assay. The reaction mixture consisted of 3 ml of a solution containing 200 ~1 of 1% o-dianisidine in 25 ml of 0.03% hydrogen peroxide and the reaction started by the addition of 100 ,uI of enzyme preparation, Activity was recorded as change in absorbance at 460 nm during 1 min using a Perkin-Elmer Mode1 402 spectrophotometer. Received August 14, 1974.
Freezing technique. Enzyme preparations, in either the acetate or phosphate buffer or water and at different protein concentrations or containing PVP at various concentrations, were frozen to -20°C. Freeze-thaw times were 40- 30-min duration, respectively. Thawing was allowed to occur at room temperature. Five freezethaw cycles were completed and the volume of enzyme preparation subjected to this treatment was 400 ,uI. Dehydration technique. While this technique has already been described in detail elsewhere (5, 6, 7, 23), it is desirable to repeat details of the method so that the process can be completely understood. A pressure membrane apparatus (5) has been used to manipulate the water potential of the enzyme preparation. (Water potential, as defined by Slatyer and Taylor (22), refers to the difference between the partial specific Gibbs free energy of water in a system compared with that of pure water. Dehydration to -2 bars is much less severe than dehydration to -10 bars, ) The pressure membrane apparatus consists of a cylindrical brass body closed at one end which contains a solid fiIter-support leading to a drain, A dialysis membrane (26/ 32) underlain by a Millpore filter (0.01 pm) is held in position over the filter support by an acrylic sleeve containing a Teflon insert which defines an effective filter area of 25mm diameter. 276
Copyright All rights
0 1975 by Academic Press, of reproduction in any form
Inc. reserved.
of
FREEZING
AND
DEHYDRATION
In the case of peroxidase, 400 pl of the enzyme preparation were added to the apparatus and a selected hydrostatic pressure applied. This increased the water potential of the system, and water and small solute molecules passed through the membrane. Equilibration was allowed to occur for 90 min when the enzyme system inside the chamber was in equiIibrium with the solution extomal to the chamber and at atmospheric pressure. The hydrostatic pressure was then released subjecting the internal enzyme phase to a tension equivalent to the applied pressure. The enzyme system was then rehydrated with 400 ,.J of the appropriate buffer, Hence, when different hydrostatic pressures were applied, the peroxidase preparation could be dehydrated to a range of water potentials, After rehydration the preparation was assayed and thus the effect of the dehydration treatment determined. Controls for this treatment consisted of subjecting an enzyme preparation to a hydrostatic pressure equivalent to the preparation being dehydrated. As reported previously with cataIase (7)) hydrostatic pressure aIone had no effect on enzyme activity, and all manipulations were conducted at 25°C. Ulfrauiolet difference-spectra. This technique, using matched tandem celIs in a Perkin-Elmer 402 double-beam spectrophotometer, was employed to determine if the freezing or dehydration treatments re-
Cycle number FIG. I. The c:ffect of freeze-thaw cycle number on the inactivation of peroxidase in different buffering systems. O-0, Phosphate; x-x, acetate; a-*, water.
OF PEROXIDASE
277
TABLE
1
PEROXIDME ACTIVITY EXPRKSWII s PEHCENT CONTROLS AFTER V+~RIOUS TREXPMENTG Treatment
OF
Dehydration to -10 bars
Freea-thaw 5 Cycles
33.2
53.0
23.4 12.0
44.0 38.0
Phosphate buffer, pH 6.4, 0.1 M Acetate buffer, pH 5.6, 0.1 Y Water
sulted in conformational protein molecules ( 12).
changes in the
Protein determination. The method of Lowry et al. ( 19) was used for all protein determinations. Polyacrylamide-gel electrophoresis. Discontinuous electrophoresis in 53% polyacrylamide gels was carried out in the Tris-glycine system of Davis (9). A current of 2 mA per tube was used and the peroxidase isozymes stained with benzidene-H302 ( 17). RESULTS
AND
DISCUSSION
It was found initially that five freezethaw cycles reduced the activity of peroxidase to a greater extent than a single cycle (Fig. 1). Dehydration of peroxidase over a range of water potentials (-4 to -15 bars) generated similar activity losses, so dehydration to -10 bars was arbitrariIy chosen to compare freezing and dehydration. A comparison of the effects of freezing and dehydration are presented in Table 1. All protein was recovered foIIowing the dehydration treatment, and it can be seen from Table 1 that the effect of dehydration was more severe than freezing. However it is interesting to observe that in both cases the presence of buffer ions protected the enzyme to some extent from inactivation. As Connor and Ashwood-Smith (4) pointed out, following the results of Lovelock (18) many people attributed the damaging effect of freezing to high salt concentrations. In the ifl vitro experiments reported in this paper
BEN DARBYSHIRE
278
*/a P v F!
FIG. 2. The effect of different concentrations of PVP on the stability of peroxidase following five freeze-thaw cycles. Symbols as for Fig. 1.
buffered enzyme preparations were less affected by dehydration and freezing than preparations in water alone. The protective effects of poIyviny1 pyrroIidone (PVP), at different concentrations, against damage due to freezing and dehydration were determined. Figure 2 indicates that a concentration of PVP of 0.01% was most effective in protecting the enzyme from freezing, At higher concentrations the protection afforded by PVP was maintained when the enzyme preparation was in water, However, in the presence of phosphate or acetate buffers, the effectiveness of PVP in preventing damage decreased. Mackenzie and Rasmussen (20) reported that at Iower temperatures PVP tended to be more hydrated. Perhaps at higher PVP concentrations, PVP and the enzyme molecules are competing for water molecules to maintain hydration,
FIG. 3. The protection of peroxidase against dehydration to -10 bars by increasing concmtrations of PVP. Symbols as for Fig. 1.
FIG. 4. The relationship between increasing protein concentration on the stability of peroxidase after five freeze-thaw cycles. Symbols as for Fig. 1.
and this results in an increased dehydration of peroxidase when frozen. The protective effect of different concentrations of PVP on dehydration of peroxidase is shown in Fig. 3. Protection increases until 1% PVP and higher concentrations did not result in reduced protection. Thus, PVP will protect peroxidase against the effects of both freezing and dehydration, but different concentrations are required to achieve maximum protection. To determine if protein-protein interactions influenced the effects of freezing and dehydration, the concentration of protein was increased from 0.4 pg/ml, as used in the above experiments, to higher values. Figure 4 indicates the effect of increasing protein concentration when peroxidase is frozen. It can be seen that in the presence of acetate or phosphate buffers, when the protein concentration is increased to between 2 and 4 pg/ml, the enzyme is no longer susceptible to freezing damage. In water, a concentration of at least 10 pg/ml was required to remove freezing damage. When peroxidase was dehydrated, increased protein concentration again protected the enzyme but a higher concentration of 20 pg/ml was required to achieve approximately 80% recovery of activity (Fig. 5). Ansevin and Lauffer (1) reported that low temperatures, low ionic concentrations
FREEZING
AND
DEHYDRATION
and IOW protein concentrations resulted in a depoIymerisation of tobacco mosaic virus protein. Later Laufkr (13) reported that when tobacco mosaic virus protein and insulin interacted to polymerize, release or exclusion of water occurred. It is suggested that a similar mechanism operates when increasing protein concentration results in the protection of peroxidase from the effects of dehydration and freezing. At higher concentrations, interaction of protein m0Iecules forming an ohgomeric protein may lead to a greater stability of the peroxidase molecules with less associated water and hence more recoverabIe activity during periods of desiccation. Ultraviolet difference-spectra did not reveal any conformation differences between the native and dehydrated or frozen preparations. It is possible that conformationaI changes following desiccation treatments may not have been detected due to the comparatively low concentrations of protein required before damage occurred, Similarly, polyacrylamide-gel electrophoresis was not a viable method to determine if particular isozymes were more sensitive to desiccation than others. This was because protein concentrations high enough to detect on gels protected the enzyme from either freezing or dehydration, It may require fluorescent probe analysis to determine if conformational changes were the cause of inactivation of peroxidase following desiccation. In addition, to determine if individual isozymes differ in their desiccation susceptibilities, it will most probably be necessary to separate the isozymes before subjecting them to freezing or dehydration. It has been reported (8) that the carbohydrate units of three fungaI enzymes protect these molecules against the adverse effects of dehydration. Although horseradish peroxidase is glycosylated (21) and the carbohydrate portion accounts for 18% by weight of the molecule, in this case no immediate indication of protection by the
a a&
OF PEROXIDASE
/ o:1 OL
279
I I, I I 20 20 ‘I LW Protwn con~cotrot~onl&g /mi )
FIG. 5. The effect
of dehydration
of peroxidase
to -10 bars at increasing concentrations of protein. Symbols as for Fig. 1. sugar moieties against freezing or dehydration is apparent. However carbohydrates may play a role as the protein concentration is increased, either directly by protection or by assisting in a polymerization mechanism. The interpretation of the damage due to freezing and dehydration remains essentially the same as reported previously (7). Water in the presence of macromolecules is structured (3, 10, 15, 24) and plays a part in maintaining the tridimensional structure of proteins (24). Water removal by freezing or dehydration results in an irreversibIe change in the molecule causing enzyme inactivation, In the results persented here higher protein concentrations may stabilize the molecules through protein-protein interactions and water release. The presence of optimal 1eveIs of PVP could reduce the vapour-pressure difference between the protein phase and the external environment ( 16). PVP may also interact directly with protein to form a more stable complex containing a reduced water content. In the case of freezing of peroxidase, where increasing concentrations of PVP reduced its protective effect, PVP may be competing for water molecules and result in further dehydration of the enzyme. Thus the damage caused by both the freezing and dehydration of peroxidase ilz vitro may be interpreted as a dehydration
BEN DARBYSHIRE
280
process. Further work will be required to investigate the contribution of the ionic environment to the protection of the enzyme against desiccation and to expIain fully the protective mechanism of increasing protein concentrations,
sure
membrane
device.
Lab. Pratt.
6. Darbyshire, B., and Steer, B. T. Dehydration of macromolecules, I. Effect of dehydration-rehydration on indoleacetic acid oxidase, ribonuclease, ribulosediphosphate carboxylase and ketose-I-phosphate aldolase.
AIL&. 1. Biol. Sci. 26, 591404 SUMMARY
Explanations of the mechanism of freezing injury have included the one that freezing may result in dehydration of enzymes. This hypothesis has been examined by comparing the effects of freezing and dehydration on horseradish peroxidase. It was found that freezing and dehydration reduce the activity of peroxidase when compared with the native enzyme. Polyvinyl pyrrolidone and increased protein concentration protect the enzyme against loss of activity in both treatments, Proteinprotein interactions exclude water, and this mechanism is suggested to stabilize peroxidase and protect against desiccation. Polyvinyl pyrrolidone may protect against freezing by reducing dehydration through reduced vapor-pressure difference or may stabilize by a protein-polymer interaction. ACKNOWLEDGMENTS I thank Mrs. Lynette technical assistance and protein determinations.
Dewar far competent Mr. Leith Higgins for
REFERENCES 1. Ansevin, A. T., and Lauffer, M. A. Native tobacco mosaic virus protein of molecular weight 18,000. NUttLTe (London) 183, lf301-1602 (1959). 2. Ashwood-Smith, M. J., and Warby, C. Protective effect of low and high molecular weight compounds on the stability of catalase subjected to freezing and thawing. CTyobioEogy 9, 137-140 ( 1972). Q. Chapman, G., and McLauchlan, K. A. Orientated water in the sciatic nerve of rabbit. Nature (London) 215, 391392 (1967). 4. Connor, W., and Ashwood-Smith, M. J. Cryoprotection of mammalian cells in tissue culture with polymers; possibIe mechanisms. CqobioEogy 10, 488-496 ( 1973). 5. Darbyshire, B., and Anlezark, R. N. A simple and versatile pressure fihering and pres-
21,
554 (1972).
(1973).
7. Darbyshire, B. The influence of dehydration on catalase stability. A comparison with freezing effects. Cryobiology II, 148-151 ( 1974). 8. Darbyshire, B. The function of the carbohydrate units of three fungal enzymes in their resistance to dehydration. Plant Yhysiol. 54, 717-721 (1974). 9. Davis, B. J. Disc electrophoresis. II. Method and application to human serum proteins, Ann. N.Y. Acad. Sci. 121, 404427 (1964). 10. Grant, E. H. The structure of water neighboring proteins, peptides and amino acids as deduced from dielectric measurements. Ann. N.Y. Acad. Sci. 125, 418-427 (1965). II. Heber, U. Freezing injury in relation to loss of enzyme activities and protection against freezing. Cryobi0Z0g~ 5, 188-201 (1968). 12. Kronman, M. J., and Robbins, M. Buried and exposed groups in proteins. In “Fine Structure of Proteins and Nucleic Acids” (G. D. Fasman and S. N. Timasheff, Eds.) pp, 271-416. Dekker, New York, 1970. 13. Lauffer, M. A. Protein-protein interaction: Endothermic polymerization and biological processes. In “Proteins and Their Reactions” (H. W. Schultz and A. F. Anglemier, Eds.), pp. 87-116. Avi Publishing Company, Westport, Conn., 1964. 14. Levitt, J. “The Hardiness of Plants.” Academic Press, New York 1956. 15. Ling, G. N. Hydration of macromolecules. In “Water and Aqueous Solutions. Structure, Thermodynamics and Transport Processes” (R. A. Horne, Ed. ), pp, 683-700, WiIey-Interscience, New York, 1972. 16. Litvan, G. G. Mechanism of cryoinjury in biological systems. Cryobiology 9, 182-191. (1972). 17. Liu, E. H. A simple method for determining the relative activities of individual peroxidase isozymes in a tissue extract. A&. Biochem. 56, 149-X4 ( 1973). 18. Lovelock, J. E. The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Biochtm. Biophys. Acta 11, 28-36 (1953). 19. Lowry, 0. H., Rosebrough, N. J., Fan; A. L., and Randall, R. j. Protein measurement
FREEZING
AND
DEHYDRATION
with the FoIin phenol reagent. .I. Biol. Cham. 193, 265-275 (1951). 20. MacKenzie, A. P., and Rasmussen, 13. H. Interactions in the water-poIyviny1 pyrrolidone system at low temperatures. In “Water Structure at the Water-Polymer Interface” (H. H. G. Jelhnek, Ed.) pp. 146-172. PIenum, New York, 1972. 21. Shannon, L. M., Kay, E., and Lew, J. Y. Peroxidase isozymes from horseradish roots. I. Isolation and physical properties. J. Biol.
Chew. 241, 2166-2172 (1966).
OF PEROXIDASE
281
22. Slatyer, R. O., and Taylor, S. A. Terminology in plant and soil-water relations. Nature (London) 187, 922-924 ( 1960). 23. Steer, B. T. Dehydration of Macromolecules. II. The protective effect of certain anions on ribulosediphosphate carboxylase subjected to low water potentials in vitro. AZ&. J. Bid. Sci. 26, 1435-1442 ( 1973). 24. Tait, M. J., and Franks, F. Water ical systems. Nature (Londo~z) (1971).
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