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Cryoprotection of purified rat kidney transamidinase by polyethylene glycol
29, 511-518 (1992)
CRYOBIOLOGY
Cryoprotection
of Purified Rat Kidney Transamidinase Polyethylene Glycol
by
CALVIN D. TORMANEN Department
of Chemistry,
Central Michigan
University,
Mount Pleasant,
Michigan
48859
Polyethylene glycol is a water-soluble polymer which is widely used in the pharmaceutical, cosmetic, and chemical industries. In this study, it is shown that polyethylene glycol is an effective cryoprotectant of rat kidney transamidinase purified from both the mitochondria and cytosol. Much of the activity is lost when the purified enzyme IS frozen and thawed in sodium-potassium phosphate buffer in the absence of cryoprotectants. Polyethylene glycols with molecular weights of 4000 to 10,000 were effective cryoprotectants. However, polyethylene glycols with a molecular weight of 1000 or lower inhibited the purified enzyme. A concentration of only 0.01% polyethylene glycol4000, 8000, or 10,000 was required for complete cryoprotection. In addition to polyethylene glycol, 0.5 mM ethylenediaminetetraacetic acid was required in the phosphate buffer for complete cryoprotection. The stabilization of purified transamidinase by polyethylene glycol will facilitate characterization experiD 1992 ments designed to compare the properties of the mitochondrial and cytosolic isozymes. Academic
Press, Inc.
Polyethylene glycol (PEG) is a nontoxic pounds including sugars, amino acids, and water-soluble synthetic polymer which is methylamines also provided cryoprotection widely used in the pharmaceutical, cos- for lactate dehydrogenase (8). Transamidinase (L-arginine:glycine amimetic, and chemical industries (14). PEG is used as a fractional precipitating agent for dinotransferase, EC 2.1.4.1) has been purithe purification of proteins (4, 12, 13), and it fied from the mitochondrial and cytosolic is used as a solvent for crystallizing pro- compartments of rat kidney (19). When puteins (1.5). Recently, PEG has been co- rified transamidinase is frozen and thawed valently attached to pharmacological en- much of the activity is lost. As the concenzymes to improve their therapeutic index tration of the purified enzyme was de(5), and it has been used to enhance immu- creased, the loss in activity due to freezing noblotting sensitivity (22). and thawing increased. The addition of sePurified dilute enzymes frequently un- rum albumin to solutions of the purified endergo denaturation when the solutions are zyme provided complete protection to defrozen and thawed (18). PEG has been used naturation from freezing and thawing. in at least two instances to provide cryo- However, for many applications, it would protection for proteins in solution. PEG 400 be desirable to find a cryoprotectant be(average molecular weight) protected im- sides a protein to stabilize the purified enmunoglobulins from denaturation during zyme. freezing and thawing (20). PEG 600 proA systematic study of the effect of varivided cryoprotection for purified lactate de- ous molecular weights and concentrations hydrogenase (8). A variety of other com- of PEG on the cryoprotection of a purified enzyme has not been reported in the literature. In this report, PEGS of various molecular weights and concentrations were studReceived July 1, 1991; accepted January 13, 1992. 511 001l-2240/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
CAL”IN De -,-._ .I...-.. 1 UKMANbN
512
ied to determine if they would provide cryoprotection for purified mitochondrial and cytosolic transamidinase. MATERIALS
AND
purified enzyme was stored at 4°C under a nitrogen atmosphere. Assay of transamidinase. Transamidinase was assayed as described by Van Pilsum et al. (21) with modifications (19). One unit of activity is the formation of 1 prnol L-ornithineih at 37°C. The protein concentration was determined by the method of Bradford (6) using bovine serum albumin as the standard. Effect of PEG on transamidinase activity. The effect of PEGS of various molecular weights and concentrations on the activity of purified mitochondrial and cytosolic transamidinase was studied using enzyme which had never been frozen. The protein concentration of the purified enzyme was adjusted to 0.036 mgi ml by dilution with 0.10 M sodium-potassium phosphate buffer, pH 7.4, immediately prior to the addition of PEG. The volume of transamidinase used for each assay was 0.125 ml. An aliquot of 0.125 ml of PEG dissolved in PE buffer (0.10 M sodium-potassium phosphate, 1.0 mM EDTA, pH 7.4) was added just prior to the addition of substrate to start the reaction. The remainder of the assay was performed as described above.
METHODS
Materials. Bovine serum albumin, PEG 400, PEG 600, PEG 1000, PEG 8000, and PEG 10,000 were obtained from Sigma Chemical Co. (St. Louis, MO). PEG 4000 was obtained from EM Reagents (Ann Arbor, MI). Ethylenediaminetetraacetic acid (EDTA) was purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ). Solubilization
and purification
of transamidinase.
Male Sprague-Dawley rats were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). The rats were fed Purina rat chow and had access to water ad libitum. The lights were turned on at 8 AM and off at 8 PM and the animals (approximately 500 g body wt) were sacrificed by decapitation between 9 AM and 10 AM. Cytosolic and mitochondrial transamidinase was extracted from fresh rat kidney as described previously (19). The cytosolic and mitochondrial isozymes were purified separately using the same procedure (19). The
Cryoprotection
transamidinase
by PEG.
1
I
I
I
of purified
The cryoprotective effect of PEG of various molecular weights and concentrations was studied on purified
. I.60 -
0
+/
I
I
I
0.01
0.10
1.00
PEG
Concentration
.~
I 10.0
(%I
FIG. 1. The effect of different concentrations of PEG on the activity of purified rat kidney mito-
chondrial transamidinase. Transamidinase was assayed in the presence of PEG 400 (o), PEG 600 (Cl), PEG 1000 (A), PEG 4000 (o), PEG 8000 (W), and PEG 10,000 (A) as described under materials and methods.
CRYOPROTECTION
BY
POLYETHYLENE
activity of purified mitochondrial and cytosolic rat kidney transamidinase which had never been frozen is shown in Figs. 1 and 2. PEG 400, 600, and 1000 inhibited the activity of the purified enzyme from both the mitochondria and cytosol. At a PEG 400, 600, and 1000concentration of lo%, both of the isozymes were inhibited approximately 50%. However, PEG 4000, 8000, and 10,000 activated purified mitochondrial and cytosolic isozymes which had never been frozen (Figs. 1 and 2). Both isozymes were activated approximately 10% at concentrations of PEG 4000, 8000, and 10,000 from 0.01 to 10%. The effect of different concentrations of PEG of various molecular weights on the stability of frozen-thawed purified mitochondrial and cytosolic transamidinase was studied. PEG 4000, 8000, and 10,000 provided complete cryoprotection for both isozymes at all concentrations from 0.01 to 10% (Figs. 3 and 4).
mitochondrial and cytosolic transamidinase. Purified enzyme which had never been frozen was diluted to 0.036 mg/ml with 0.10 M sodium-potassium phosphate buffer, pH 7.4, immediately prior to the addition of PEG. The volume of enzyme was 0.125 ml, and the volume of PEG in PE buffer was also 0.125 ml. The samples were in 13 x loo-mm glass test tubes, and they were stoppered after adding a nitrogen atmosphere. The samples were stored overnight in a freezer at -20°C. The following day, the test tubes were removed from the freezer and were placed in a water bath at room temperature until the samples were thawed. Immediately after thawing, the transamidinase activity was determined as described previously. Each sample contained 4.5 x 10e6g of enzyme. Effect of omitting EDTA on cryoprotection by PEG. The effect of freezing and thawing purified mitochondrial and cytosolic transamidinase in the absence of EDTA was studied. The experiments were performed exactly as described above except that only PEG 10,000 was used and no EDTA was present in the phosphate buffer. RESULTS
The effect of different concentrations of PEG of various molecular weights on the
Oo-
/
513
GLYCOL
I
I
I
I
0.01
0.10
1.00
10.0
PEG Concentration
(%)
FIG. 2. The effect of different concentrations of PEG on the activity of purified cytosolic trans-
amidinase. The enzyme was assayed in the presence of PEG 400 (o), PEG 600 (Cl), PEG 1000(A), PEG 4000 (o), PEG 8000 (m), and PEG 10,000 (A) as described in the text.
1
514
CALVIN D. TORMANEN
0.M
0
-4/ 0
I
I
0 01
010
I
PEG Concentration
I
(%)
FIG. 3. Recovery of purified mitochondrial transamidinase activity after storage in the freezer. The enzyme was stored at - 20°C overnight under a N, atmosphere in the presence of different concentrations of PEG. The samples were thawed and the transamidinase activity was assayed in the presence of PEG 400 (o), PEG 600 (O), PEG 1000(A), PEG 4000 (O), PEG 8000 (W), and PEG 10,ooO(A) as described in the text.
The minimum concentration of PEG required for cryoprotection was determined. For both purified mitochondrial and cytosolic transamidinase, PEG 10,000 provided only partial cryoprotection at 0.001% in results not shown. A concentration of 0.0001% PEG 10,000 provided no cryoprotection for either isozyme. The effect of the absence of EDTA in the phosphate buffer used for cryoprotection is shown in Fig. 5. Purified mitochondrial transamidinase which had never been frozen had about 10% lower activity when assayed in the absence of EDTA. When the enzyme was frozen and thawed without EDTA present, only 44% of the activity was recovered compared to 66% when frozen and thawed in the presence of EDTA. Similar results were obtained with purified cytosolic enzyme. When purified mito-
chondrial transamidinase was frozen and thawed in the absence of EDTA with PEG 10,000 present, only partial cryoprotection was provided by 0.01 to 10% PEG (Fig. 5). Similar results were obtained with purified cytosolic enzyme. DISCUSSION
Carpenter and Crowe (8) and Arakawa et al. (2) have proposed that PEG stabilizes proteins by preferential exclusion. The PEG is sterically hindered from making contact with the protein’s surface. The thermodynamic consequences of preferential solute exclusion causes stabilization of the protein during freezing and thawing. The larger molecular weight PEGS would have greater steric hindrance. PEG with a molecular weight of 4000 and greater provided complete cryoprotection for purified
CRYOPROTECTION
BY POLYETHYLENE
515
GLYCOL
0.60
I
I
0.10
1.00
PEG Concentration
I 10.0
(%I
FIG. 4. Recovery of purified cytosolic transamidinase activity after storage in the freezer. The enzyme was stored at - 20°C overnight under a N, atmosphere in the presence of different concentrations of PEG. The samples were thawed and the enzyme activity was assayed in the presence of PEG 400 (O), PEG 600 (O), PEG 1000 (A), PEG 4000 (o), PEG 8000 (W), and PEG 10,000 (A) as described in the text.
transamidinase (Figs. 3 and 4) while PEG with a molecular weight of 1000 and lower inhibited the enzyme (Figs. 1 and 2). Ingham (12) has shown that the effectiveness of PEG as a protein-precipitating agent depends on the molecular weight of the polymer. PEGS with a molecular weight of 4000 and 6000 were very effective in precipitating proteins while PEGS with a molecular weight of 600 and 1000 precipitated very little protein. Thus, the effect of the molecular weight of PEG on the precipitation of proteins (12) agrees with the results shown in Figs. 3 and 4 for cryoprotection of purified transamidinase by PEG. PEG with a molecular weight of 4000 or greater is most effective for both protein precipitation and protein cryoprotection. The concentration of PEG required to precipitate proteins is from 5 to 20% (12).
However, the concentration of PEG 4000, 8000, and 10,000 required for cryoprotection of purified mitochondrial and cytosolic transamidinase was only 0.01% (1.0 x 10e5 A4 for PEG 10,000) as shown in Figs. 3 and 4. The concentration of transamidinase used for the cryoprotection experiments was 2.0 x lo-’ M. Therefore, only 50 molecules of PEG 10,000 were required per molecule of enzyme for complete cryoprotection. Carpenter and Crowe (8) have reported that PEG 600 provided complete cryoprotection of lactate dehydrogenase at concentrations from 1.5% (25 mM) to 60% (1 .O A!). The use of PEG 600 at a concentration of less than 1.5% or PEGS of different molecular weights was not reported. The activity of purified transamidinase which had never been frozen was decreased by the presence of PEG 400, 600,
516
CALVIN D. TORMANEN
. 0
0.0001
0.01
0.001
PEG 10,000
Concentration
0.10
1.00
10.0
(461
FIG. 5. Cryoprotection of purified mitochondrial transamidinase by PEG 10,000in the presence or absence of EDTA. Fresh control with EDTA (o), frozen-thawed with EDTA (O), fresh control without EDTA (A), and frozen-thawed without EDTA (A) as described in the text.
and 1000 in the assay (Figs. 1 and 2). The served that enzymes are stabilized by ininhibition may be due to the increased in- creasing the concentration of the enzyme or fluence of the terminal hydroxyl groups in by adding another protein such as bovine the low molecular weight PEGS. In results serum albumin (8, 18). The presence of EDTA at 0.5 rnM was not shown, glycerol at a concentration of 1.0 M inhibited purified transamidinase by required for the complete cryoprotection of purified transamidinase by PEG 10,000 16%. In contrast, the activity of purified trans- (Fig. 5). This was unexpected since EDTA amidinase which had never been frozen is not required in the assay or for storage of was increased by the presence of PEG the enzyme at 4°C. Also, transamidinase is 4000, 8000, and 10,000 (Figs. 1 and 2). In not inhibited by the presence of low conresults not shown, purified transamidinase centrations of divalent cations (results not was activated 20% by the presence of 0.10 shown). No divalent cations were added to mg/ml bovine serum albumin in the assay the buffers used in these experiments. Carsolution. Also, in results not shown, the penter et al. (7) observed that the cryoprotection of phosphofructokinase by organic specific activity of both the mitochondrial and cytosolic purified enzyme increased solutes was enhanced by the presence of when the concentration of the enzyme in divalent cations. Also, Hazen et al. (9) have the assay was above 0.018 mg/ml, and the reported that the cryoprotection of antibodspecific activity of purified transamidinase ies by organic solutes was enhanced by the decreased when the enzyme concentration presence of divalent cations. The effect of was below 0.018 mg/ml. It is commonly ob- divalent cations on the cryoprotection of
CRYOPROTECTION
BY
POLYETHYLENE
purified transamidinase will be studied in future work. Normalization of conditions when conducting cryoprotection experiments is important since purified enzymes are very sensitive to changes in environment (18). All of the cryoprotection experiments described in this report were conducted using standardized conditions for cooling, storage, and thawing. Glass test tubes were used throughout since purified transamidinase was also denatured when frozen and thawed in plastic test tubes (results not shown). When sodium-potassium phosphate buffer is frozen, the pH drops from 7.4 to about 4 (10, 11, 17). In results not shown, PEG 10,000 at a concentration of 10% prevented the drop in pH of phosphate buffer when frozen. However, PEG 10,000 at concentrations of 1% or lower did not prevent the drop in pH. Since PEG 10,000 provided complete cryoprotection for transamidinase at concentrations as low as O.Ol%, the stabilization is not due to a pH effect. The stabilization of purified transamidinase by PEG will facilitate further characterization experiments designed to compare the properties of the mitochondrial and cytosolic isozymes. The results of the experiments with PEG described in this report do not show any qualitative differences between the mitochondrial and cytosolic isozymes. In a previously reported study, the specific activity was the only property in which the isozymes were found to differ (19). The subunit molecular mass, the electrophoretic mobility under nondenaturing conditions, and the activation energy were similar. The lower specific activity of the cytosolic isozyme compared to the mitochondrial isozyme can also be seen by comparing the enzyme activities shown in Fig. 2 with Fig. 1. Freeze denaturation of enzymes is a serious problem in the biotechnology industry. Since PEG is nontoxic, biodegradable, water-soluble, inexpensive, and nondena-
517
GLYCOL
turing, the use of PEG as a protein cryoprotectant should be explored further. However, because the surface properties of proteins varies (1, S), PEG may not provide cryoprotection to all proteins. Also, PEG can decrease the stability of proteins at high temperatures because of increased hydrophobic interactions (1, 3, 16). ACKNOWLEDGMENT
This work was supported by a grant from the Michigan Polymer Consortium (22270). REFERENCES
1. Arakawa, T., and Timasheff, S. N. Mechanism of poly(ethylene glycol) interaction with proteins. Biochemistry 24, 67X-6762 (1985). 2. Arakawa, T., Carpenter, J. F., Kita, Y. A., and Crowe, J. H. The basis for toxicity of certain cryoprotectants: A hypothesis. Cryobiology 27, 401415 (1990). 3. Arakawa, T., Kita, Y., and Carpenter, J. F. Protein-solvent interactions in pharmaceutical formulations. Pharm. Res. 8, 285-291 (1991). 4. Atha, D. H., and Ingham, K. C. Mechanism of precipitation of proteins by polyethylene gly~01s.J. Biol. Chem. 256, 12,108-12,117 (1981). 5. Banci. L., Bertini, I., Caliceti, P., Monsu’ Scolaro, L., Schiavon, O., and Veronese, F. M. Spectroscopic characterization of polyethyleneglycol modified superoxide dismutase: ‘H NMR studies on its Cu,Co, derivative. J. Znorg. Biochem. 39, 149-159 (1990). 6. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 (1976). 7. 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. Eiochem. Biophys. 250, 505512 (1986). 8. Carpenter, J. F., and Crowe, J. H. The mechanism of cryoprotection of proteins by solutes. Cryobiology
25, 24255
(1988).
9. Hazen, K. C., Bourgeois, L. D., and Carpenter, J. F. Cryoprotection of antibody by organic solutes and organic solute/divalent cation mixtures. Arch. Biochem. Biophys. 267, 363-371 (1988). 10. Hill, J. P., and Dickinson, F. M. Enzyme storage-to freeze or not to freeze? Biochem. Sot. Trans. 17, 1079-1080
(1989).
I I. Hill, J. P., and Buckley, P. D. The use of pH in-
518
12.
13.
14.
15.
16.
17.
CAL”IN D* ----. -_--1 UKMANbN
dicators to identify suitable environments for freezing samples in aqueous and mixed aqueouslnonaqueous solutions. Anal. Biochem. 192, 3.58361 (1991). Ingham, K. C. Polyethylene glycol in aqueous solution: solvent perturbation and gel filtration studies. Arch. Biochem. Biophys. 184, 59-68 (1977). Ingham, K. C. Precipitation of proteins with polyethylene glycol. In “Methods in Enzymology” (M. P. Deutscher, Ed.), pp. 301-306. Academic Press, San Diego, 1990. Kawai, F. The biochemistry of degradation of polyethers. CRC Crir. Rev. Biotech. 6, 273-306 (1987). Lee, J. C., and Lee, L. L. Y. Preferential solvent interactions between proteins and polyethylene glycols. J. Biol. Chem. 256, 625-631 (1981). Lee, L. L., and Lee, J. C. Thermal stability of proteins in the presence of poly(ethylene gly~01s).Biochemistry 26, 7813-7819 (1987). Seguro K., Tamiya, T., Tsuchiya, T., and Matsumoto, J. J. Cryoprotective effect of sodium glutamate and lysine-HCI on freeze denaturation
of lactate debydrogenase. Cryobiology 21, 7079 (1990). 18. Tamiya, T., Okahashi, N., Sakuma, R., Aoyama, T., Akahane, T., and Matsumoto, J. J. Freeze denaturation of enzymes and its prevention with additives. Cryobiology 22, 44fS.456 (1985). 19. Tormanen, C. D. Comparison of the properties of purified mitochondrial and cytosolic rat kidney transamidinase. Int. J. Biochem. 22, 1243-1250 (1990). 20. Tsutsayeva, A. A., Pushkar, N. S., Markova, V. M., Itkin, Y. A., Markovsky, A. L., Gavrilova, I. I., Ivanov, L. V., Moiseyev, V. A., and Medvedyeva, G. G. The mechanism of action of low temperature and cryoprotective 15, agents on immunoproteins. Cryobiology 403-lO7 ( 1978). 21. Van Pilsum, J. F., Taylor, D., Zakis, B., and McCormick, P. Simplified assay for transamidinase activities of rat kidney transamidinase. Anal. Biochem. 35, 277-286 (1970). 22. Zeng, C., Suzuki, Y., and Alpert, E. Polyethylene glycol significantly enhances the transfer of membrane immunoblotting. Anal. Biochem. 189, 197-201 (1990).
CRYOBIOLOGY
Cryoprotection
of Purified Rat Kidney Transamidinase Polyethylene Glycol
by
CALVIN D. TORMANEN Department
of Chemistry,
Central Michigan
University,
Mount Pleasant,
Michigan
48859
Polyethylene glycol is a water-soluble polymer which is widely used in the pharmaceutical, cosmetic, and chemical industries. In this study, it is shown that polyethylene glycol is an effective cryoprotectant of rat kidney transamidinase purified from both the mitochondria and cytosol. Much of the activity is lost when the purified enzyme IS frozen and thawed in sodium-potassium phosphate buffer in the absence of cryoprotectants. Polyethylene glycols with molecular weights of 4000 to 10,000 were effective cryoprotectants. However, polyethylene glycols with a molecular weight of 1000 or lower inhibited the purified enzyme. A concentration of only 0.01% polyethylene glycol4000, 8000, or 10,000 was required for complete cryoprotection. In addition to polyethylene glycol, 0.5 mM ethylenediaminetetraacetic acid was required in the phosphate buffer for complete cryoprotection. The stabilization of purified transamidinase by polyethylene glycol will facilitate characterization experiD 1992 ments designed to compare the properties of the mitochondrial and cytosolic isozymes. Academic
Press, Inc.
Polyethylene glycol (PEG) is a nontoxic pounds including sugars, amino acids, and water-soluble synthetic polymer which is methylamines also provided cryoprotection widely used in the pharmaceutical, cos- for lactate dehydrogenase (8). Transamidinase (L-arginine:glycine amimetic, and chemical industries (14). PEG is used as a fractional precipitating agent for dinotransferase, EC 2.1.4.1) has been purithe purification of proteins (4, 12, 13), and it fied from the mitochondrial and cytosolic is used as a solvent for crystallizing pro- compartments of rat kidney (19). When puteins (1.5). Recently, PEG has been co- rified transamidinase is frozen and thawed valently attached to pharmacological en- much of the activity is lost. As the concenzymes to improve their therapeutic index tration of the purified enzyme was de(5), and it has been used to enhance immu- creased, the loss in activity due to freezing noblotting sensitivity (22). and thawing increased. The addition of sePurified dilute enzymes frequently un- rum albumin to solutions of the purified endergo denaturation when the solutions are zyme provided complete protection to defrozen and thawed (18). PEG has been used naturation from freezing and thawing. in at least two instances to provide cryo- However, for many applications, it would protection for proteins in solution. PEG 400 be desirable to find a cryoprotectant be(average molecular weight) protected im- sides a protein to stabilize the purified enmunoglobulins from denaturation during zyme. freezing and thawing (20). PEG 600 proA systematic study of the effect of varivided cryoprotection for purified lactate de- ous molecular weights and concentrations hydrogenase (8). A variety of other com- of PEG on the cryoprotection of a purified enzyme has not been reported in the literature. In this report, PEGS of various molecular weights and concentrations were studReceived July 1, 1991; accepted January 13, 1992. 511 001l-2240/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
CAL”IN De -,-._ .I...-.. 1 UKMANbN
512
ied to determine if they would provide cryoprotection for purified mitochondrial and cytosolic transamidinase. MATERIALS
AND
purified enzyme was stored at 4°C under a nitrogen atmosphere. Assay of transamidinase. Transamidinase was assayed as described by Van Pilsum et al. (21) with modifications (19). One unit of activity is the formation of 1 prnol L-ornithineih at 37°C. The protein concentration was determined by the method of Bradford (6) using bovine serum albumin as the standard. Effect of PEG on transamidinase activity. The effect of PEGS of various molecular weights and concentrations on the activity of purified mitochondrial and cytosolic transamidinase was studied using enzyme which had never been frozen. The protein concentration of the purified enzyme was adjusted to 0.036 mgi ml by dilution with 0.10 M sodium-potassium phosphate buffer, pH 7.4, immediately prior to the addition of PEG. The volume of transamidinase used for each assay was 0.125 ml. An aliquot of 0.125 ml of PEG dissolved in PE buffer (0.10 M sodium-potassium phosphate, 1.0 mM EDTA, pH 7.4) was added just prior to the addition of substrate to start the reaction. The remainder of the assay was performed as described above.
METHODS
Materials. Bovine serum albumin, PEG 400, PEG 600, PEG 1000, PEG 8000, and PEG 10,000 were obtained from Sigma Chemical Co. (St. Louis, MO). PEG 4000 was obtained from EM Reagents (Ann Arbor, MI). Ethylenediaminetetraacetic acid (EDTA) was purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ). Solubilization
and purification
of transamidinase.
Male Sprague-Dawley rats were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). The rats were fed Purina rat chow and had access to water ad libitum. The lights were turned on at 8 AM and off at 8 PM and the animals (approximately 500 g body wt) were sacrificed by decapitation between 9 AM and 10 AM. Cytosolic and mitochondrial transamidinase was extracted from fresh rat kidney as described previously (19). The cytosolic and mitochondrial isozymes were purified separately using the same procedure (19). The
Cryoprotection
transamidinase
by PEG.
1
I
I
I
of purified
The cryoprotective effect of PEG of various molecular weights and concentrations was studied on purified
. I.60 -
0
+/
I
I
I
0.01
0.10
1.00
PEG
Concentration
.~
I 10.0
(%I
FIG. 1. The effect of different concentrations of PEG on the activity of purified rat kidney mito-
chondrial transamidinase. Transamidinase was assayed in the presence of PEG 400 (o), PEG 600 (Cl), PEG 1000 (A), PEG 4000 (o), PEG 8000 (W), and PEG 10,000 (A) as described under materials and methods.
CRYOPROTECTION
BY
POLYETHYLENE
activity of purified mitochondrial and cytosolic rat kidney transamidinase which had never been frozen is shown in Figs. 1 and 2. PEG 400, 600, and 1000 inhibited the activity of the purified enzyme from both the mitochondria and cytosol. At a PEG 400, 600, and 1000concentration of lo%, both of the isozymes were inhibited approximately 50%. However, PEG 4000, 8000, and 10,000 activated purified mitochondrial and cytosolic isozymes which had never been frozen (Figs. 1 and 2). Both isozymes were activated approximately 10% at concentrations of PEG 4000, 8000, and 10,000 from 0.01 to 10%. The effect of different concentrations of PEG of various molecular weights on the stability of frozen-thawed purified mitochondrial and cytosolic transamidinase was studied. PEG 4000, 8000, and 10,000 provided complete cryoprotection for both isozymes at all concentrations from 0.01 to 10% (Figs. 3 and 4).
mitochondrial and cytosolic transamidinase. Purified enzyme which had never been frozen was diluted to 0.036 mg/ml with 0.10 M sodium-potassium phosphate buffer, pH 7.4, immediately prior to the addition of PEG. The volume of enzyme was 0.125 ml, and the volume of PEG in PE buffer was also 0.125 ml. The samples were in 13 x loo-mm glass test tubes, and they were stoppered after adding a nitrogen atmosphere. The samples were stored overnight in a freezer at -20°C. The following day, the test tubes were removed from the freezer and were placed in a water bath at room temperature until the samples were thawed. Immediately after thawing, the transamidinase activity was determined as described previously. Each sample contained 4.5 x 10e6g of enzyme. Effect of omitting EDTA on cryoprotection by PEG. The effect of freezing and thawing purified mitochondrial and cytosolic transamidinase in the absence of EDTA was studied. The experiments were performed exactly as described above except that only PEG 10,000 was used and no EDTA was present in the phosphate buffer. RESULTS
The effect of different concentrations of PEG of various molecular weights on the
Oo-
/
513
GLYCOL
I
I
I
I
0.01
0.10
1.00
10.0
PEG Concentration
(%)
FIG. 2. The effect of different concentrations of PEG on the activity of purified cytosolic trans-
amidinase. The enzyme was assayed in the presence of PEG 400 (o), PEG 600 (Cl), PEG 1000(A), PEG 4000 (o), PEG 8000 (m), and PEG 10,000 (A) as described in the text.
1
514
CALVIN D. TORMANEN
0.M
0
-4/ 0
I
I
0 01
010
I
PEG Concentration
I
(%)
FIG. 3. Recovery of purified mitochondrial transamidinase activity after storage in the freezer. The enzyme was stored at - 20°C overnight under a N, atmosphere in the presence of different concentrations of PEG. The samples were thawed and the transamidinase activity was assayed in the presence of PEG 400 (o), PEG 600 (O), PEG 1000(A), PEG 4000 (O), PEG 8000 (W), and PEG 10,ooO(A) as described in the text.
The minimum concentration of PEG required for cryoprotection was determined. For both purified mitochondrial and cytosolic transamidinase, PEG 10,000 provided only partial cryoprotection at 0.001% in results not shown. A concentration of 0.0001% PEG 10,000 provided no cryoprotection for either isozyme. The effect of the absence of EDTA in the phosphate buffer used for cryoprotection is shown in Fig. 5. Purified mitochondrial transamidinase which had never been frozen had about 10% lower activity when assayed in the absence of EDTA. When the enzyme was frozen and thawed without EDTA present, only 44% of the activity was recovered compared to 66% when frozen and thawed in the presence of EDTA. Similar results were obtained with purified cytosolic enzyme. When purified mito-
chondrial transamidinase was frozen and thawed in the absence of EDTA with PEG 10,000 present, only partial cryoprotection was provided by 0.01 to 10% PEG (Fig. 5). Similar results were obtained with purified cytosolic enzyme. DISCUSSION
Carpenter and Crowe (8) and Arakawa et al. (2) have proposed that PEG stabilizes proteins by preferential exclusion. The PEG is sterically hindered from making contact with the protein’s surface. The thermodynamic consequences of preferential solute exclusion causes stabilization of the protein during freezing and thawing. The larger molecular weight PEGS would have greater steric hindrance. PEG with a molecular weight of 4000 and greater provided complete cryoprotection for purified
CRYOPROTECTION
BY POLYETHYLENE
515
GLYCOL
0.60
I
I
0.10
1.00
PEG Concentration
I 10.0
(%I
FIG. 4. Recovery of purified cytosolic transamidinase activity after storage in the freezer. The enzyme was stored at - 20°C overnight under a N, atmosphere in the presence of different concentrations of PEG. The samples were thawed and the enzyme activity was assayed in the presence of PEG 400 (O), PEG 600 (O), PEG 1000 (A), PEG 4000 (o), PEG 8000 (W), and PEG 10,000 (A) as described in the text.
transamidinase (Figs. 3 and 4) while PEG with a molecular weight of 1000 and lower inhibited the enzyme (Figs. 1 and 2). Ingham (12) has shown that the effectiveness of PEG as a protein-precipitating agent depends on the molecular weight of the polymer. PEGS with a molecular weight of 4000 and 6000 were very effective in precipitating proteins while PEGS with a molecular weight of 600 and 1000 precipitated very little protein. Thus, the effect of the molecular weight of PEG on the precipitation of proteins (12) agrees with the results shown in Figs. 3 and 4 for cryoprotection of purified transamidinase by PEG. PEG with a molecular weight of 4000 or greater is most effective for both protein precipitation and protein cryoprotection. The concentration of PEG required to precipitate proteins is from 5 to 20% (12).
However, the concentration of PEG 4000, 8000, and 10,000 required for cryoprotection of purified mitochondrial and cytosolic transamidinase was only 0.01% (1.0 x 10e5 A4 for PEG 10,000) as shown in Figs. 3 and 4. The concentration of transamidinase used for the cryoprotection experiments was 2.0 x lo-’ M. Therefore, only 50 molecules of PEG 10,000 were required per molecule of enzyme for complete cryoprotection. Carpenter and Crowe (8) have reported that PEG 600 provided complete cryoprotection of lactate dehydrogenase at concentrations from 1.5% (25 mM) to 60% (1 .O A!). The use of PEG 600 at a concentration of less than 1.5% or PEGS of different molecular weights was not reported. The activity of purified transamidinase which had never been frozen was decreased by the presence of PEG 400, 600,
516
CALVIN D. TORMANEN
. 0
0.0001
0.01
0.001
PEG 10,000
Concentration
0.10
1.00
10.0
(461
FIG. 5. Cryoprotection of purified mitochondrial transamidinase by PEG 10,000in the presence or absence of EDTA. Fresh control with EDTA (o), frozen-thawed with EDTA (O), fresh control without EDTA (A), and frozen-thawed without EDTA (A) as described in the text.
and 1000 in the assay (Figs. 1 and 2). The served that enzymes are stabilized by ininhibition may be due to the increased in- creasing the concentration of the enzyme or fluence of the terminal hydroxyl groups in by adding another protein such as bovine the low molecular weight PEGS. In results serum albumin (8, 18). The presence of EDTA at 0.5 rnM was not shown, glycerol at a concentration of 1.0 M inhibited purified transamidinase by required for the complete cryoprotection of purified transamidinase by PEG 10,000 16%. In contrast, the activity of purified trans- (Fig. 5). This was unexpected since EDTA amidinase which had never been frozen is not required in the assay or for storage of was increased by the presence of PEG the enzyme at 4°C. Also, transamidinase is 4000, 8000, and 10,000 (Figs. 1 and 2). In not inhibited by the presence of low conresults not shown, purified transamidinase centrations of divalent cations (results not was activated 20% by the presence of 0.10 shown). No divalent cations were added to mg/ml bovine serum albumin in the assay the buffers used in these experiments. Carsolution. Also, in results not shown, the penter et al. (7) observed that the cryoprotection of phosphofructokinase by organic specific activity of both the mitochondrial and cytosolic purified enzyme increased solutes was enhanced by the presence of when the concentration of the enzyme in divalent cations. Also, Hazen et al. (9) have the assay was above 0.018 mg/ml, and the reported that the cryoprotection of antibodspecific activity of purified transamidinase ies by organic solutes was enhanced by the decreased when the enzyme concentration presence of divalent cations. The effect of was below 0.018 mg/ml. It is commonly ob- divalent cations on the cryoprotection of
CRYOPROTECTION
BY
POLYETHYLENE
purified transamidinase will be studied in future work. Normalization of conditions when conducting cryoprotection experiments is important since purified enzymes are very sensitive to changes in environment (18). All of the cryoprotection experiments described in this report were conducted using standardized conditions for cooling, storage, and thawing. Glass test tubes were used throughout since purified transamidinase was also denatured when frozen and thawed in plastic test tubes (results not shown). When sodium-potassium phosphate buffer is frozen, the pH drops from 7.4 to about 4 (10, 11, 17). In results not shown, PEG 10,000 at a concentration of 10% prevented the drop in pH of phosphate buffer when frozen. However, PEG 10,000 at concentrations of 1% or lower did not prevent the drop in pH. Since PEG 10,000 provided complete cryoprotection for transamidinase at concentrations as low as O.Ol%, the stabilization is not due to a pH effect. The stabilization of purified transamidinase by PEG will facilitate further characterization experiments designed to compare the properties of the mitochondrial and cytosolic isozymes. The results of the experiments with PEG described in this report do not show any qualitative differences between the mitochondrial and cytosolic isozymes. In a previously reported study, the specific activity was the only property in which the isozymes were found to differ (19). The subunit molecular mass, the electrophoretic mobility under nondenaturing conditions, and the activation energy were similar. The lower specific activity of the cytosolic isozyme compared to the mitochondrial isozyme can also be seen by comparing the enzyme activities shown in Fig. 2 with Fig. 1. Freeze denaturation of enzymes is a serious problem in the biotechnology industry. Since PEG is nontoxic, biodegradable, water-soluble, inexpensive, and nondena-
517
GLYCOL
turing, the use of PEG as a protein cryoprotectant should be explored further. However, because the surface properties of proteins varies (1, S), PEG may not provide cryoprotection to all proteins. Also, PEG can decrease the stability of proteins at high temperatures because of increased hydrophobic interactions (1, 3, 16). ACKNOWLEDGMENT
This work was supported by a grant from the Michigan Polymer Consortium (22270). REFERENCES
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