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Organochemistry of cryoinjury and cryophylaxis 3. Dissociation of a pure protein by cryoprotectants: The basic mechanism of toxicity?
312
ANNUAL
MEETING
adopted one or more of them as a replacement for the previously exclusively used bicarbonate/CO2 system. A general consensus of opinion is that the buffers exhibit little, if any, toxicity and that effects observed in their presence are not related to any specific action of the compound other than the control of pH. However, Elford and Walter [Nature (London) New Biol. 236, 58 (1972)] have used two of these buffers in potassium-rich bathing media and have shown improvements over other buffers in the function and morphology of smooth muscle cooled to -79°C in the unfrozen state. The experiments described here provide evidence for a protective effect of the compounds when used with Chinese hamster cells cooled to -196°C and preserved in the frozen state. A positive correlation exists between the relative efficacy of the buffers as cryoprotective agents and their relative ability to stabilize the three-dimensional conformation of macromolecules. A further positive correlation exists between cryoprotective qualities and buffering capacity as expressed by the term ApK,. The results will be discussed in the light of the possible role of the buffers in influencing the structure of water in the vicinity of the cells during the freezing and thawing process. (Supported in part by National Institutes of Health Grant 1 ROl GM17959-OlAl). 20. Organochemistry of Cryoinjury and Cryophy1axi.s 5. Dissociation of a Pure Protein by Cryoprotectants: The Basic Mechanism of Toxicity? W. N. FISHBEIN (Biochemistry Branch. Armed
Forces Institute 20305).
of Pathology, Washington, DC
Jackbean urease can be reliably crystallized and purified in a stable form with MW 480,000. Under appropriate conditions it can be dissociated to halfunits (MW 240,000) which retain full activity. and subsequently to inactive subunits (MW ~10,000), which, in the absence of SDS, spontaneously aggregate to inactive high polymers. The forms can be separated, identified, and quantitated by acrylamide gel electrophoresis, providing a convenient test system to evaluate cryoprotectants as dissociating agents. At concentrations of 30-90% (v/v), all of the cryophylactics studied, produced some dissocia.tion of the enzyme. By evaluating the concentration of agent, time of reaction, residual enzyme activity, and molecular forms, families of cryoprotectants could be graded in decreasing order
SYMPOSIUM
ABSTRACTS
of potency as dissociating agents. For glycols. ethylene glycol > 1,2-propanediol > 1, a-butanediol > 2,3-butanediol > 1,3-propanediol > 1,3butanediol Z 1,4-butanediol. Clearly vicinal OH groups provide greater dissociating potency, as they do for cryophylactic potency (Abstract 61), and molecular size is also critical. For alcohols: isopropanol > set-butanol > methanol > tertbutanol > ethanol 2 n-propanol. Thus shielded OH groups are more effective than exposed groups, until a certain molecular size is exceeded; aside from this, the smaller the compound, the more potent it is. The data suggest that the compounds react directly with the protein, and, in fact, fit into hydrophobic clefts in the macromolecule. The pattern for N-substituted-amides is similar to those cited, and DMSO is also quite effective as a dissociating agent. Although high concentrations are involved, similar levels are produced when solutions are frozen with the multimolar cryoprotectant levels commonly assumed to be essential for cryophylaxis (Abstract 16). Macromolecular dissociation may therefore be the basic mechanism of toxicity for monomeric cryophylactic agents. by Lipase in Frozen Sys21. Lipid Hydrolysis tems. L. G. PARDUCCI AND 0. FENNEMA (Department of Food Science, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI 53706).
It is well-known that cert,ain enzyme-catalyzed reactions proceed in frozen systems at relatively low temperatures. How initial reaction rates and over-all courses of these reactions are influenced by the freezing treatment and by temperature of frozen storage remains to be clearly defined. The effect of various conditions of freezing and frozen storage on the course of hydrolysis of tributyrin by lipase has been investigated in a simple liquid system. Initial rates of hydrolysis were measured at a number of different temperatures, and extents of the reactions were monitored during continued storage at these temperatures. A measure of lipase activity was obtained by automatic titration of fatty acids released during hydrolysis, after a simple extraction procedure. It appears that not only initial reaction velocity, but also the ultimate FXtent of hydrolysis in a given system is influenced significantly by the storage temperature. Observed behavior is similar to that which occurs in unfrozen low-moisture systems.
2: THE ROLE OF WATER BIOLOGICAL SYSTEMS
22. The Influence of Water at Biological Interfaces. W. DROST-HANSEN* (Department of Chem-
IN
istry, Laborabory for Water Research, University of Miami, Coral Gables, FL 33124).
ANNUAL
MEETING
adopted one or more of them as a replacement for the previously exclusively used bicarbonate/CO2 system. A general consensus of opinion is that the buffers exhibit little, if any, toxicity and that effects observed in their presence are not related to any specific action of the compound other than the control of pH. However, Elford and Walter [Nature (London) New Biol. 236, 58 (1972)] have used two of these buffers in potassium-rich bathing media and have shown improvements over other buffers in the function and morphology of smooth muscle cooled to -79°C in the unfrozen state. The experiments described here provide evidence for a protective effect of the compounds when used with Chinese hamster cells cooled to -196°C and preserved in the frozen state. A positive correlation exists between the relative efficacy of the buffers as cryoprotective agents and their relative ability to stabilize the three-dimensional conformation of macromolecules. A further positive correlation exists between cryoprotective qualities and buffering capacity as expressed by the term ApK,. The results will be discussed in the light of the possible role of the buffers in influencing the structure of water in the vicinity of the cells during the freezing and thawing process. (Supported in part by National Institutes of Health Grant 1 ROl GM17959-OlAl). 20. Organochemistry of Cryoinjury and Cryophy1axi.s 5. Dissociation of a Pure Protein by Cryoprotectants: The Basic Mechanism of Toxicity? W. N. FISHBEIN (Biochemistry Branch. Armed
Forces Institute 20305).
of Pathology, Washington, DC
Jackbean urease can be reliably crystallized and purified in a stable form with MW 480,000. Under appropriate conditions it can be dissociated to halfunits (MW 240,000) which retain full activity. and subsequently to inactive subunits (MW ~10,000), which, in the absence of SDS, spontaneously aggregate to inactive high polymers. The forms can be separated, identified, and quantitated by acrylamide gel electrophoresis, providing a convenient test system to evaluate cryoprotectants as dissociating agents. At concentrations of 30-90% (v/v), all of the cryophylactics studied, produced some dissocia.tion of the enzyme. By evaluating the concentration of agent, time of reaction, residual enzyme activity, and molecular forms, families of cryoprotectants could be graded in decreasing order
SYMPOSIUM
ABSTRACTS
of potency as dissociating agents. For glycols. ethylene glycol > 1,2-propanediol > 1, a-butanediol > 2,3-butanediol > 1,3-propanediol > 1,3butanediol Z 1,4-butanediol. Clearly vicinal OH groups provide greater dissociating potency, as they do for cryophylactic potency (Abstract 61), and molecular size is also critical. For alcohols: isopropanol > set-butanol > methanol > tertbutanol > ethanol 2 n-propanol. Thus shielded OH groups are more effective than exposed groups, until a certain molecular size is exceeded; aside from this, the smaller the compound, the more potent it is. The data suggest that the compounds react directly with the protein, and, in fact, fit into hydrophobic clefts in the macromolecule. The pattern for N-substituted-amides is similar to those cited, and DMSO is also quite effective as a dissociating agent. Although high concentrations are involved, similar levels are produced when solutions are frozen with the multimolar cryoprotectant levels commonly assumed to be essential for cryophylaxis (Abstract 16). Macromolecular dissociation may therefore be the basic mechanism of toxicity for monomeric cryophylactic agents. by Lipase in Frozen Sys21. Lipid Hydrolysis tems. L. G. PARDUCCI AND 0. FENNEMA (Department of Food Science, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI 53706).
It is well-known that cert,ain enzyme-catalyzed reactions proceed in frozen systems at relatively low temperatures. How initial reaction rates and over-all courses of these reactions are influenced by the freezing treatment and by temperature of frozen storage remains to be clearly defined. The effect of various conditions of freezing and frozen storage on the course of hydrolysis of tributyrin by lipase has been investigated in a simple liquid system. Initial rates of hydrolysis were measured at a number of different temperatures, and extents of the reactions were monitored during continued storage at these temperatures. A measure of lipase activity was obtained by automatic titration of fatty acids released during hydrolysis, after a simple extraction procedure. It appears that not only initial reaction velocity, but also the ultimate FXtent of hydrolysis in a given system is influenced significantly by the storage temperature. Observed behavior is similar to that which occurs in unfrozen low-moisture systems.
2: THE ROLE OF WATER BIOLOGICAL SYSTEMS
22. The Influence of Water at Biological Interfaces. W. DROST-HANSEN* (Department of Chem-
IN
istry, Laborabory for Water Research, University of Miami, Coral Gables, FL 33124).