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zinc mixtures
CRYOBIOLOGY
25, 372-376 (1988)
BRIEF COMMUNICATION Long-Term Preservation of Dried Phosphofructokinase and Sugar/Zinc Mixtures
by Sugars
JOHN F. CARPENTER,* BETH MARTIN,* STEPHEN H. LOOMIS,*,? AND JOHN H. CROWE* *Department of Zoology, University of California, Davis, California 95616; and TDepartment of Zoology, Connecticut College, New London, Connecticut 06320 We have demonstrated that sugars and suger/zinc mixtures can be used to preserve the activity of dried phosphofructokinase (PFK) during long-term storage over CaS04. After 9 weeks in the presence of either 200 m&f sucrose or 200 mM trehalose little loss of PFK activity was noted, with almost 60% of the original prefreeze-dry activity recovered when samples were rehydrated. Even reducing sugars protected the dried enzyme throughout the entire storage period. Of the sugars tested, 200 r&14lactose provided the most stability to PFK; at the end of the dry storage, over 80% of the initial activity was recovered. With either 200 mM maltose or 400 mM glucose, about 40% of the initial activity was recovered at the end of the experiment. With all the sugars tested, the addition of 0.6 mM Zn*+ to sugar/PFK mixtures enhanced the stability of the enzyme, and no long-term adverse effects of the metal ion on enzyme activity were noted. 0 1988 Academic press, 1~.
Sugars stabilize labile proteins during freeze-drying and rehydration (reviewed in (lo)), including even the extremely labile enzyme phosphofructokinase (PFK), which is completely and irreversibly inactivated when it is frozen or dried in the absence of sugars (U). However, in previous studies PFK was held in the dried state for only 1 day (4, 6, 10). We now show that this enzyme can be kept dry for at least several weeks without loss of activity. There were three main reasons for our concern about the interaction of sugars with biological materials during long periods of dehydration. (a) A common characteristic of organisms that can survive removal of essentially all of their intracellular water is the presence of high concentrations (often in excess of 20% of their dry weight) of nonreducing sugars such as sucrose or trehalose (2,7, 13,21-23). Many of these organisms can survive in the dried Received January 14, 1988; accepted February 22, 1988.
state for several years (8,9,22). Therefore, to substantiate further the proposal (6) that one role of these sugars is to preserve labile proteins in dehydration-tolerant organisms, it is necessary to demonstrate that the stability afforded by sucrose and trehalose to a dried protein is maintained during longterm storage. (b) Many of the sugars (e.g., maltose, glucose, and lactose) that have been used to stabilize dried proteins during short-term experiments are reducing sugars. These sugars have a propensity to participate in protein browning via the Maillard reaction, a process that usually takes several days to become detectable (12, 15). Thus, from a practical viewpoint it is important to know whether storage conditions can be defined under which reducing sugars can be used to stabilize dried proteins, without loss of catalytic activity due to protein browning. (c) Finally, in earlier studies we found that the addition of divalent zinc to a PFK/sugar solution greatly enhanced the recovery of PFK activity after freezeor air-drying (4, 6). In order for this phe-
372 OOll-2240/88 $3.00 Copyright 6 1988 by Academic Press, Inc. All tights of reproduction in any form reserved.
BRIEF
nomenon to be used to advantage in the preparation of dried enzyme/sugar mixtures for storage, it must be established that there are no adverse effects induced by the metal ion with time. MATERIALS
AND
373
COMMUNICATION
METHODS
Materials. Glycerol-3-phosphate dehydrogenase (rabbit muscle) and triosephosphate isomerase (rabbit muscle) were products of Boehringer-Mannheim. Phosphofructokinase (type III) and aldolase (type I) were purchased from Sigma Chemical Co. Trehalose, sucrose, lactose, and maltose were obtained from Pfanstiehl Laboratories, and glucose was purchased from Mallinckrodt . Purification and assay of phosphofructokinase. Rabbit skeletal muscle PFK, ob-
tained commercially as a crystalline suspension in ammonium sulfate, was purified and stored following the procedures described by Carpenter et al. (5). PFK activity was assayed at 25°C using the fructose1,6-bisphosphate-coupled assay described by Bock and Frieden (3) and Carpenter et al. (5). Protein was assayed according to the method of Peterson (20). Freeze-drying and storage of phosphofructokinase. Prior to each experiment,
PFK was dialyzed (4°C) for several hours against 1 mM sodium borate buffer (pH 7.8 at 23°C) containing 25 mit4 K,SO,, 2 mM (NH&S04, and 5 mM dithiothreitol. To avoid complications due to enzyme adsorption to glass, freeze-drying and storage of PFK were performed in polypropylene Eppendorf test tubes. An aliquot of the enzyme stock was added to the appropriate solution of sugar (prepared in the above buffer) to give a final PFK concentration of 150 &ml and the desired sugar concentration; 200 mM for trehalose, sucrose, maltose, and lactose, and 400 mA4 for glucose. PFK/sugar mixtures were also prepared with 0.6 mM ZnSO,. Any effects of this addition are attributed to zinc and not the anion because the buffer already contains
27 mM sulfate. Catalytic activity of each mixture was assayed, and then several identical sample tubes were prepared, each containing 75 ~1 of the mixture. All samples of a given mixture were frozen simultaneously by immersion in liquid nitrogen for 90 sec. The frozen samples were then lyophilized for 20 hr on a VirTis lyophilizer. The dried samples were stored in desiccators over CaSO,. At time intervals throughout the storage period, triplicate samples were removed, rehydrated with 5 mM dithiothreitol (pH 7.8 at 23”C), and assayed for residual PFK activity. The results are expressed as the percentage of prefreeze-dry activity recovered after rehydration, corrected for any differences between the volumes of rehydrated and pretreated samples. RESULTS
AND
DISCUSSION
Long-term storage of dried PFK with nonreducing sugars. As can be seen in Fig.
1, even after 9 weeks of storage in the presence of either 200 mM sucrose or 200 mM trehalose there is little loss of PFK activity. At the end of the experiment, after rehydration, almost 60% of the original prefreezedry activity is recovered with either sugar. The critical role that these disaccharides play in the natural history of dehydrationtolerant organisms is emphasized by the fact that in several cases survival of desiccation has been shown to correlate directly with the level of sugar in the organism (11, 16, 18, 21). Furthermore, it is known that these organisms can remain viable after many years in the dried state (8, 9, 22), and the present results indicate that the stabilizing influence of sugars on proteins could be maintained during long periods of time. It is not as apparent, however, whether transition metals could play a role in preserving proteins in vivo in dried organisms. The results in Fig. 1 demonstrate that the addition of 0.6 mM Zn*+ to PFK/ disaccharide mixtures has no adverse effect on the long-term preservation of PFK cat-
374
BRIEF
COMMUNICATION
FIG. 1. Activity of freeze-dried phosphofructokinase recovered after storage over CaSO,and rehydration. Samples were prepared in 200 tnM trehalose (A) alone, 200 m&f sucrose (B) alone (O), and with the addition of 0.6 mM ZnSO, (m). Data points represent the means & SD for three independent dried samples. Where vertical bars are absent, the SD is less than the size of the symbol.
alytic activity. Even though it is not as apparent at the relatively high sugar concentrations employed in the present study, our earlier experiments showed that zinc enhanced the protection afforded to dried PFK by lower concentrations of sugars (4, 6). It is attractive to speculate that zinc could serve to increase the stability of labile proteins in dehydration-tolerant organisms, but this may not be a reasonable suggestion since it is known that several enzymes are inhibited in vitro by transition metals (1, 6). However, from a practical viewpoint, for enzymes that are not specifically inhibited by these ions it could be advantageous to use divalent cations to enhance stability of the dried proteins. Long-term storage of dried PFK with reducing sugars. As is the case with sucrose and trehalose, the catalytic activity of PFK dried in the presence of maltose, lactose, or glucose is only slightly diminished after 9 weeks of storage, and most of the activity loss occurs during the first week (Fig. 2). Of
FIG. 2. Activity of freeze-dried phosphofructokinase recovered after storage over CaSO, and rehydration. Samples were prepared in 200 n&I lactose (A) alone, 200 mM maltose (B) atone, 400 mM glucose (C) alone (a), and with the addition of 0.6 mMZnS0, (m). pointsrepresent the means 2 SD for three independent dried samples, Where vertical bars are absent, the SD is less than the size of the symbol.
Data
the sugars tested, 200 mM lactose provides the most stability to PFK; at the end of the dry storage, over 80% of the initial activity is recovered after the enzyme is rehydrated (Fig. 2A). With either 200 mM maltose or 400 mM glucose, about 40% of the initial activity is recovered at the end of the experiment (Figs. 2B and 2C). These results are somewhat surprising since reducing sugars are known to participate in protein browning at reduced water activities (12, 15). Browning occurs via a Maillard reaction between primary amines (e.g., those in lysine residues) and the carbony1 group on the sugar (12). This sort of reaction, especially in its earlier stages, does not necessarily lead to protein unfold-
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375
COMMUNICATION
ing. For example, it has been demonstrated with ovalbumin that reaction of the protein’s primary amine groups with glucose can be detected before more extensive protein browning leads to denaturation (14). With PFK it seems unlikely that such an event could occur without loss of activity because it has been shown that covalently linking pyridoxal5’-phosphate to the lysine residues in this enzyme can lead to complete inactivation (17). Thus, since PFK catalytic activity is maintained for the duration of our experiments, it appears that protein browning is not operative under the storage conditions used in the present study. One potential explanation for this finding is that the samples held over CaSO, are actually too dry to foster reaction of the sugars’ carbonyl groups to the protein’s primary amines. To promote protein browning, samples are often maintained at a slightly higher hydration. For example, Loomis et al. (15) found that at 60% relative humidity bovine serum albumin was browned in the presence of glucose. Similarly, a browning reaction of glucose with ovalbumin was seen at 65% relative humidity (14). Mouradian et al. (19) found that samples of sarcoplasmic reticulum vesicles freeze-dried with glucose and stored at 35% relative humidity showed rapid and extensive browning, whereas only minimal browning was noted at 11% relative humidity. Therefore, it appears that whenever circumstances dictate the need to use a reducing sugar to stabilize a dried protein (e.g., 200 mM lactose was the most effective stabilizer in the present study), the optimal means of storage is to keep samples at 0% relative humidity over a desiccant. In Fig. 2, it can be seen that under these conditions the addition of 0.6 mA4ZnS04 to PFK/reducing sugar mixtures also is not deleterious to PFK activity during longterm storage. However, in earlier research on ovalbumin/glucose mixtures stored at 65% relative humidity, it was found that
cu*+
and Fe3+ accelerated the browning reaction and the denaturation of the protein (14). Presumably, another transition metal such as Zn2+ could be expected to display the same property when the residual moisture was at this level (cf. (14)). Thus, in conjunction with the above arguments regarding the importance of hydration to sample stability, it can be suggested that storage of dried protein samples over CaSO, is also the method of choice to avoid damage potentially induced by divalent cations. In conclusion, we have demonstrated that sugars and sugar/zinc mixtures can be used to preserve the activity of dried phosphofructokinase during long-term storage. The finding that trehalose and sucrose, two disaccharides commonly found in dehydration-tolerant organisms, display this property provides further support for the contention (6) that these sugars can serve to protect labile proteins in organisms that can survive lengthy bouts of desiccation. From a practical viewpoint, we have demonstrated that even reducing sugars can be used to preserve a dried protein if the samples are kept over CaS04. Finally, it appears that, when appropriate, divalent zinc can be added to sugar/enzyme mixtures to enhance enzyme stability, without any detrimental effects appearing during long-term dry storage. ACKNOWLEDGMENT
This research was supported by National Science Foundation Grant DMB 8.5-18194. REFERENCES
1. Barman, T. E. “Enzyme Handbook,” SpringerVerlag, New York, 1969. 2. Bewley, J. D. Physiological aspects of desiccation tolerance. Annu. Rev. Plant Physiol. 30, 195238 (1979). 3. Bock, P. E., and Frieden, C. pH-Induced cold lability of rabbit skeletal muscle phosphofructokinase. Biochemistry 13, 4191-4196 (1974). 4. Carpenter, J. F., Crowe, L. M., and Crowe, J. H. Stabilization of phosphofructokinase with sugars during freeze-drying: Characterization of
376
5.
6.
7.
8.
9. 10.
11.
12.
13.
BRIEF COMMUNICATION
enhanced protection in the presence of divalent cations. Biochim. Biophys. Acta 923, 109-115 (1987). Carpenter, J. F., Hand, S. C., Crowe, L. M., and Crowe, J. H. Cryoprotection of phosphofructokinase with organic solutes: Characterization of enhanced protection in the presence of divalent cations. Arch. Biochem. Biophys. 250,505512 (1986). Carpenter, J. F., Martin, B., Crowe, L. M., and Crowe, J. H. Stabilization of phosphofructokinase during air-drying with sugars and sugar/ transition metal mixtures. Cryobiology 24,455464 (1987). Clegg, J. S. The origin of trehalose and its significance during the formation of encysted dormant embryos of Artemia salina. Camp. Biothem. Physiol. 14, 135-143 (1965). Crowe, J. H., and Clegg, J. S. “Anhydrobiosis.” Dowden, Hutchinson, and Ross, Stroudsburg, PA, 1973. Crowe, J. H., and Clegg, J. S. “Dry Biological Systems.” Academic Press, New York, 1978. Crowe, J. H., Crowe, L. M., Carpenter, J. F., and Aurell Wistrom, C. Stabilization of dry phospholipid bilayers and proteins by sugars. Biochem. .I. 242, l-10 (1987). Crowe, J. H., Madin, K. A. C., and Loomis, S. H. Anhydrobiosis in nematodes: Metabolism during resumption of activity. J. Exp. 2001. 201, 57-64 (1977). Feeney, R. E., Blankenhorn, G., and Dixon, H. B. F. Carbonyl-amine reactions in protein chemistry. Adv. Protein Chem. 29, 135-203 (1975). Hoekstra, F. A. Water content in relation to stress in pollen. In “Membranes, Metabolism, and Dry Organisms” (A. C. Leopold, Ed.), pp. 102-122. Comstock, Ithaca, NY, 1986.
14. Kato, Y., Watanabe, K., and Sato, Y. Effect of some metals on the Maillard reaction of ovalbumin. J. Agric. Food Chem. 29, 540-543 (1981). 15. Loomis, S. H., O’Dell, S. J., and Crowe, J. H. Anhydrobiosis in nematodes: Inhibition of the browning reaction of reducing sugars with dry proteins. .I. Exp. 2001. 208, 355-360 (1979). 16. Madin, K. A. C., and Crowe, J. H. Anhydrobiosis in nematodes: Carbohydrate and lipid metabolism during dehydration. .I. Exp. Zool. 193, 335-342 (1975). 17. Mansour, T. E. Phosphofructokinase. Curr. Top. Cell. Regul. 5, 146 (1972). 18. Martin, M. C., Diaz, L. A., Manzanal, M. B., and Hardisson, C. Role of trehalose in the spores of Streptomyces.
19.
20.
21.
22. 23.
FEBS Microbial.
Lett. 35,49-54
(1986). Mouradian, R., Womersley, C., Crowe, L. M., and Crowe, J. H. Degradation of functional integrity during long-term storage of a freezedried biological membrane. Cryobiology 22, 119-127 (1985). Peterson, G. L. A simplification of the protein assay method of Lowry et al. which is generally more applicable. Anal. Biochem. 83, 346-356 (1977). Van Laere, A., Francois, A., Overloop, K., Verbeke, M., and Van Gerven, L. Relation between germination, trehalose and the status of water in Phycomycetes blakesleeanus spores as measured by proton-NMR. J. Gen. Microbial. 133, 239-245 (1987). Womersley, C. Biochemical and physiological aspects of anhydrobiosis. Comp. Biochem. Physiol. 7OB, 669-678 (1981). Womersley, C., and Smith L. Anhydrobiosis in nematodes. I. The role of glycerol, myo-inositol and trehalose during desiccation. Comp. Biothem. Physiol. 7OB, 579-586 (1981).
25, 372-376 (1988)
BRIEF COMMUNICATION Long-Term Preservation of Dried Phosphofructokinase and Sugar/Zinc Mixtures
by Sugars
JOHN F. CARPENTER,* BETH MARTIN,* STEPHEN H. LOOMIS,*,? AND JOHN H. CROWE* *Department of Zoology, University of California, Davis, California 95616; and TDepartment of Zoology, Connecticut College, New London, Connecticut 06320 We have demonstrated that sugars and suger/zinc mixtures can be used to preserve the activity of dried phosphofructokinase (PFK) during long-term storage over CaS04. After 9 weeks in the presence of either 200 m&f sucrose or 200 mM trehalose little loss of PFK activity was noted, with almost 60% of the original prefreeze-dry activity recovered when samples were rehydrated. Even reducing sugars protected the dried enzyme throughout the entire storage period. Of the sugars tested, 200 r&14lactose provided the most stability to PFK; at the end of the dry storage, over 80% of the initial activity was recovered. With either 200 mM maltose or 400 mM glucose, about 40% of the initial activity was recovered at the end of the experiment. With all the sugars tested, the addition of 0.6 mM Zn*+ to sugar/PFK mixtures enhanced the stability of the enzyme, and no long-term adverse effects of the metal ion on enzyme activity were noted. 0 1988 Academic press, 1~.
Sugars stabilize labile proteins during freeze-drying and rehydration (reviewed in (lo)), including even the extremely labile enzyme phosphofructokinase (PFK), which is completely and irreversibly inactivated when it is frozen or dried in the absence of sugars (U). However, in previous studies PFK was held in the dried state for only 1 day (4, 6, 10). We now show that this enzyme can be kept dry for at least several weeks without loss of activity. There were three main reasons for our concern about the interaction of sugars with biological materials during long periods of dehydration. (a) A common characteristic of organisms that can survive removal of essentially all of their intracellular water is the presence of high concentrations (often in excess of 20% of their dry weight) of nonreducing sugars such as sucrose or trehalose (2,7, 13,21-23). Many of these organisms can survive in the dried Received January 14, 1988; accepted February 22, 1988.
state for several years (8,9,22). Therefore, to substantiate further the proposal (6) that one role of these sugars is to preserve labile proteins in dehydration-tolerant organisms, it is necessary to demonstrate that the stability afforded by sucrose and trehalose to a dried protein is maintained during longterm storage. (b) Many of the sugars (e.g., maltose, glucose, and lactose) that have been used to stabilize dried proteins during short-term experiments are reducing sugars. These sugars have a propensity to participate in protein browning via the Maillard reaction, a process that usually takes several days to become detectable (12, 15). Thus, from a practical viewpoint it is important to know whether storage conditions can be defined under which reducing sugars can be used to stabilize dried proteins, without loss of catalytic activity due to protein browning. (c) Finally, in earlier studies we found that the addition of divalent zinc to a PFK/sugar solution greatly enhanced the recovery of PFK activity after freezeor air-drying (4, 6). In order for this phe-
372 OOll-2240/88 $3.00 Copyright 6 1988 by Academic Press, Inc. All tights of reproduction in any form reserved.
BRIEF
nomenon to be used to advantage in the preparation of dried enzyme/sugar mixtures for storage, it must be established that there are no adverse effects induced by the metal ion with time. MATERIALS
AND
373
COMMUNICATION
METHODS
Materials. Glycerol-3-phosphate dehydrogenase (rabbit muscle) and triosephosphate isomerase (rabbit muscle) were products of Boehringer-Mannheim. Phosphofructokinase (type III) and aldolase (type I) were purchased from Sigma Chemical Co. Trehalose, sucrose, lactose, and maltose were obtained from Pfanstiehl Laboratories, and glucose was purchased from Mallinckrodt . Purification and assay of phosphofructokinase. Rabbit skeletal muscle PFK, ob-
tained commercially as a crystalline suspension in ammonium sulfate, was purified and stored following the procedures described by Carpenter et al. (5). PFK activity was assayed at 25°C using the fructose1,6-bisphosphate-coupled assay described by Bock and Frieden (3) and Carpenter et al. (5). Protein was assayed according to the method of Peterson (20). Freeze-drying and storage of phosphofructokinase. Prior to each experiment,
PFK was dialyzed (4°C) for several hours against 1 mM sodium borate buffer (pH 7.8 at 23°C) containing 25 mit4 K,SO,, 2 mM (NH&S04, and 5 mM dithiothreitol. To avoid complications due to enzyme adsorption to glass, freeze-drying and storage of PFK were performed in polypropylene Eppendorf test tubes. An aliquot of the enzyme stock was added to the appropriate solution of sugar (prepared in the above buffer) to give a final PFK concentration of 150 &ml and the desired sugar concentration; 200 mM for trehalose, sucrose, maltose, and lactose, and 400 mA4 for glucose. PFK/sugar mixtures were also prepared with 0.6 mM ZnSO,. Any effects of this addition are attributed to zinc and not the anion because the buffer already contains
27 mM sulfate. Catalytic activity of each mixture was assayed, and then several identical sample tubes were prepared, each containing 75 ~1 of the mixture. All samples of a given mixture were frozen simultaneously by immersion in liquid nitrogen for 90 sec. The frozen samples were then lyophilized for 20 hr on a VirTis lyophilizer. The dried samples were stored in desiccators over CaSO,. At time intervals throughout the storage period, triplicate samples were removed, rehydrated with 5 mM dithiothreitol (pH 7.8 at 23”C), and assayed for residual PFK activity. The results are expressed as the percentage of prefreeze-dry activity recovered after rehydration, corrected for any differences between the volumes of rehydrated and pretreated samples. RESULTS
AND
DISCUSSION
Long-term storage of dried PFK with nonreducing sugars. As can be seen in Fig.
1, even after 9 weeks of storage in the presence of either 200 mM sucrose or 200 mM trehalose there is little loss of PFK activity. At the end of the experiment, after rehydration, almost 60% of the original prefreezedry activity is recovered with either sugar. The critical role that these disaccharides play in the natural history of dehydrationtolerant organisms is emphasized by the fact that in several cases survival of desiccation has been shown to correlate directly with the level of sugar in the organism (11, 16, 18, 21). Furthermore, it is known that these organisms can remain viable after many years in the dried state (8, 9, 22), and the present results indicate that the stabilizing influence of sugars on proteins could be maintained during long periods of time. It is not as apparent, however, whether transition metals could play a role in preserving proteins in vivo in dried organisms. The results in Fig. 1 demonstrate that the addition of 0.6 mM Zn*+ to PFK/ disaccharide mixtures has no adverse effect on the long-term preservation of PFK cat-
374
BRIEF
COMMUNICATION
FIG. 1. Activity of freeze-dried phosphofructokinase recovered after storage over CaSO,and rehydration. Samples were prepared in 200 tnM trehalose (A) alone, 200 m&f sucrose (B) alone (O), and with the addition of 0.6 mM ZnSO, (m). Data points represent the means & SD for three independent dried samples. Where vertical bars are absent, the SD is less than the size of the symbol.
alytic activity. Even though it is not as apparent at the relatively high sugar concentrations employed in the present study, our earlier experiments showed that zinc enhanced the protection afforded to dried PFK by lower concentrations of sugars (4, 6). It is attractive to speculate that zinc could serve to increase the stability of labile proteins in dehydration-tolerant organisms, but this may not be a reasonable suggestion since it is known that several enzymes are inhibited in vitro by transition metals (1, 6). However, from a practical viewpoint, for enzymes that are not specifically inhibited by these ions it could be advantageous to use divalent cations to enhance stability of the dried proteins. Long-term storage of dried PFK with reducing sugars. As is the case with sucrose and trehalose, the catalytic activity of PFK dried in the presence of maltose, lactose, or glucose is only slightly diminished after 9 weeks of storage, and most of the activity loss occurs during the first week (Fig. 2). Of
FIG. 2. Activity of freeze-dried phosphofructokinase recovered after storage over CaSO, and rehydration. Samples were prepared in 200 n&I lactose (A) alone, 200 mM maltose (B) atone, 400 mM glucose (C) alone (a), and with the addition of 0.6 mMZnS0, (m). pointsrepresent the means 2 SD for three independent dried samples, Where vertical bars are absent, the SD is less than the size of the symbol.
Data
the sugars tested, 200 mM lactose provides the most stability to PFK; at the end of the dry storage, over 80% of the initial activity is recovered after the enzyme is rehydrated (Fig. 2A). With either 200 mM maltose or 400 mM glucose, about 40% of the initial activity is recovered at the end of the experiment (Figs. 2B and 2C). These results are somewhat surprising since reducing sugars are known to participate in protein browning at reduced water activities (12, 15). Browning occurs via a Maillard reaction between primary amines (e.g., those in lysine residues) and the carbony1 group on the sugar (12). This sort of reaction, especially in its earlier stages, does not necessarily lead to protein unfold-
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375
COMMUNICATION
ing. For example, it has been demonstrated with ovalbumin that reaction of the protein’s primary amine groups with glucose can be detected before more extensive protein browning leads to denaturation (14). With PFK it seems unlikely that such an event could occur without loss of activity because it has been shown that covalently linking pyridoxal5’-phosphate to the lysine residues in this enzyme can lead to complete inactivation (17). Thus, since PFK catalytic activity is maintained for the duration of our experiments, it appears that protein browning is not operative under the storage conditions used in the present study. One potential explanation for this finding is that the samples held over CaSO, are actually too dry to foster reaction of the sugars’ carbonyl groups to the protein’s primary amines. To promote protein browning, samples are often maintained at a slightly higher hydration. For example, Loomis et al. (15) found that at 60% relative humidity bovine serum albumin was browned in the presence of glucose. Similarly, a browning reaction of glucose with ovalbumin was seen at 65% relative humidity (14). Mouradian et al. (19) found that samples of sarcoplasmic reticulum vesicles freeze-dried with glucose and stored at 35% relative humidity showed rapid and extensive browning, whereas only minimal browning was noted at 11% relative humidity. Therefore, it appears that whenever circumstances dictate the need to use a reducing sugar to stabilize a dried protein (e.g., 200 mM lactose was the most effective stabilizer in the present study), the optimal means of storage is to keep samples at 0% relative humidity over a desiccant. In Fig. 2, it can be seen that under these conditions the addition of 0.6 mA4ZnS04 to PFK/reducing sugar mixtures also is not deleterious to PFK activity during longterm storage. However, in earlier research on ovalbumin/glucose mixtures stored at 65% relative humidity, it was found that
cu*+
and Fe3+ accelerated the browning reaction and the denaturation of the protein (14). Presumably, another transition metal such as Zn2+ could be expected to display the same property when the residual moisture was at this level (cf. (14)). Thus, in conjunction with the above arguments regarding the importance of hydration to sample stability, it can be suggested that storage of dried protein samples over CaSO, is also the method of choice to avoid damage potentially induced by divalent cations. In conclusion, we have demonstrated that sugars and sugar/zinc mixtures can be used to preserve the activity of dried phosphofructokinase during long-term storage. The finding that trehalose and sucrose, two disaccharides commonly found in dehydration-tolerant organisms, display this property provides further support for the contention (6) that these sugars can serve to protect labile proteins in organisms that can survive lengthy bouts of desiccation. From a practical viewpoint, we have demonstrated that even reducing sugars can be used to preserve a dried protein if the samples are kept over CaS04. Finally, it appears that, when appropriate, divalent zinc can be added to sugar/enzyme mixtures to enhance enzyme stability, without any detrimental effects appearing during long-term dry storage. ACKNOWLEDGMENT
This research was supported by National Science Foundation Grant DMB 8.5-18194. REFERENCES
1. Barman, T. E. “Enzyme Handbook,” SpringerVerlag, New York, 1969. 2. Bewley, J. D. Physiological aspects of desiccation tolerance. Annu. Rev. Plant Physiol. 30, 195238 (1979). 3. Bock, P. E., and Frieden, C. pH-Induced cold lability of rabbit skeletal muscle phosphofructokinase. Biochemistry 13, 4191-4196 (1974). 4. Carpenter, J. F., Crowe, L. M., and Crowe, J. H. Stabilization of phosphofructokinase with sugars during freeze-drying: Characterization of
376
5.
6.
7.
8.
9. 10.
11.
12.
13.
BRIEF COMMUNICATION
enhanced protection in the presence of divalent cations. Biochim. Biophys. Acta 923, 109-115 (1987). Carpenter, J. F., Hand, S. C., Crowe, L. M., and Crowe, J. H. Cryoprotection of phosphofructokinase with organic solutes: Characterization of enhanced protection in the presence of divalent cations. Arch. Biochem. Biophys. 250,505512 (1986). Carpenter, J. F., Martin, B., Crowe, L. M., and Crowe, J. H. Stabilization of phosphofructokinase during air-drying with sugars and sugar/ transition metal mixtures. Cryobiology 24,455464 (1987). Clegg, J. S. The origin of trehalose and its significance during the formation of encysted dormant embryos of Artemia salina. Camp. Biothem. Physiol. 14, 135-143 (1965). Crowe, J. H., and Clegg, J. S. “Anhydrobiosis.” Dowden, Hutchinson, and Ross, Stroudsburg, PA, 1973. Crowe, J. H., and Clegg, J. S. “Dry Biological Systems.” Academic Press, New York, 1978. Crowe, J. H., Crowe, L. M., Carpenter, J. F., and Aurell Wistrom, C. Stabilization of dry phospholipid bilayers and proteins by sugars. Biochem. .I. 242, l-10 (1987). Crowe, J. H., Madin, K. A. C., and Loomis, S. H. Anhydrobiosis in nematodes: Metabolism during resumption of activity. J. Exp. 2001. 201, 57-64 (1977). Feeney, R. E., Blankenhorn, G., and Dixon, H. B. F. Carbonyl-amine reactions in protein chemistry. Adv. Protein Chem. 29, 135-203 (1975). Hoekstra, F. A. Water content in relation to stress in pollen. In “Membranes, Metabolism, and Dry Organisms” (A. C. Leopold, Ed.), pp. 102-122. Comstock, Ithaca, NY, 1986.
14. Kato, Y., Watanabe, K., and Sato, Y. Effect of some metals on the Maillard reaction of ovalbumin. J. Agric. Food Chem. 29, 540-543 (1981). 15. Loomis, S. H., O’Dell, S. J., and Crowe, J. H. Anhydrobiosis in nematodes: Inhibition of the browning reaction of reducing sugars with dry proteins. .I. Exp. 2001. 208, 355-360 (1979). 16. Madin, K. A. C., and Crowe, J. H. Anhydrobiosis in nematodes: Carbohydrate and lipid metabolism during dehydration. .I. Exp. Zool. 193, 335-342 (1975). 17. Mansour, T. E. Phosphofructokinase. Curr. Top. Cell. Regul. 5, 146 (1972). 18. Martin, M. C., Diaz, L. A., Manzanal, M. B., and Hardisson, C. Role of trehalose in the spores of Streptomyces.
19.
20.
21.
22. 23.
FEBS Microbial.
Lett. 35,49-54
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