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PubMed1996

Oxidative damage and antioxidants in Rana sylvatica, the freeze-tolerant wood frog.

Freeze-tolerant wood frogs (Rana sylvatica) must endure prolonged ischemia on freezing. Reperfusion on thawing brings with it the potential or oxidative damage due to reactive oxygen species formation, a well-known consequence of mammalian ischemia-reperfusion. To determine whether oxidative damage occurs during thawing and how frogs deal with this, we examined oxidative damage and antioxidant and prooxidant systems in tissues of Rana sylvatica and a nonfreezing relative, Rana pipiens. Glutathione status indicated little oxidative stress in tissues during freezing or thawing; an increase of the glutathione pool in the oxidized form was observed during freezing only in Rana sylvatica kidney (by 85%) and brain (by 33%). Oxidative damage to tissue lipids, measured as the levels of thiobarbituric acid-reactive substances and/or by an Fe(III)-xylenol orange assay, did not increase above control values pver a freeze-thaw time course. Correlative data showing increased activities of some antioxidant enzymes during freezing, notably glutathione peroxidase (increasing 1.2- to 2.5-fold), as well as constitutively higher activities of antioxidant enzymes and higher levels of glutathione in the freeze-tolerant species compared with Rana pipiens, suggest that antioxidant defenses play a key role in amphibian freeze tolerance.

Adaptation, Physiological↗
PubMed2004

Assessing pH and oxygenation in cryotherapy-induced cytotoxicity and tissue response to freezing.

The microenvironmental pH and oxygenation is known to influence tumor cell response to heat, radiation, photodynamic and even chemotherapy. We have studied the previously untested influence of acidity and hypoxia on tumor and endothelial cell sensitivity to freezing. In addition, we have measured changes in oxygenation in vivo in murine FSaII fibrosarcomas after freeze injury. A low pH or low oxygenation environment was found to increase the sensitivity of tumor and endothelial cells to freezing at -20 degrees C or -40 degrees C in vitro. However, low pH and low oxygenation combined did not further increase cryosensitivity of the cells. In vivo, tumor oxygenation after freeze injury was studied immediately or 1-3 days after a standard freezing protocol was applied to FSaII tumors ranging from 250-500 mm3 grown in the rear-limb of C3H mice. Tumor oxygenation at the edge of the iceball was found to transiently increase 1-2 hours after freezing. At 1-3 days after freezing, a treatment that delayed FSaII tumor growth by approximately 1.5-fold, the mean tumor oxygenation was significantly increased by up to 2.5-fold from a control level of 5 mmHg partial pressure of oxygen (pO2), especially at the periphery of the tumor. We conclude that manipulation of pH or oxygenation has potential to increase the anti-tumor effects of minimally invasive cryosurgical techniques. Furthermore, the dynamic changes in oxygenation after freeze injury in vivo suggests value in combining cryotherapy with treatments dependent on oxygenation levels. Ultimately, these may be routes to more reliable treatment response with fewer recurrences.

Animals↗
PubMed2006

Heat and mass transfer scale-up issues during freeze-drying, III: control and characterization of dryer differences via operational qualification tests.

The objective of this research was to estimate differences in heat and mass transfer between freeze dryers due to inherent design characteristics using data obtained from sublimation tests. This study also aimed to provide guidelines for convenient scale-up of the freeze-drying process. Data obtained from sublimation tests performed on laboratory-scale, pilot, and production freeze dryers were used to evaluate various heat and mass transfer parameters: nonuniformity in shelf surface temperatures, resistance of pipe, refrigeration system, and condenser. Emissivity measurements of relevant surfaces such as the chamber wall and the freeze dryer door were taken to evaluate the impact of atypical radiation heat transfer during scale-up. "Hot" and "cold" spots were identified on the shelf surface of different freeze dryers, and the impact of variation in shelf surface temperatures on the primary drying time and the product temperature during primary drying was studied. Calculations performed using emissivity measurements on different freeze dryers suggest that a front vial in the laboratory lyophilizer received 1.8 times more heat than a front vial in a manufacturing freeze dryer operating at a shelf temperature of -25 degrees C and a chamber pressure of 150 mTorr during primary drying. Therefore, front vials in the laboratory are much more atypical than front vials in manufacturing. Steady-state heat and mass transfer equations were used to study a combination of different scale-up issues pertinent during lyophilization cycles commonly used for the freeze-drying of pharmaceuticals.

Chemistry, Pharmaceutical↗
PubMed1993

Glucose concentration regulates freeze tolerance in the wood frog Rana sylvatica.

In spring, the lowest temperature during freezing that can be survived by wood frogs (Rana sylvatica) from southern Ohio is approximately -3 degrees C. We investigated whether the thermal limit of freeze tolerance in these frogs is regulated by tissue levels of glucose, a putative cryoprotectant that is distributed to tissues during freezing. Frogs receiving exogenous glucose injections prior to freezing showed dose-dependent increases in glucose within the heart, liver, skeletal muscle and blood. Tissue glucose concentrations were further elevated during freezing by the production of endogenous glucose. Most glucose-loaded frogs survived freezing to -5 degrees C, whereas all control (saline-injected) frogs succumbed. Further, we investigated some mechanisms by which glucose might function as a cryoprotectant in R. sylvatica. Organ dehydration, a normal, beneficial response that reduces freezing injury to tissues, occurred independently of tissue glucose concentrations. However, elevated glucose levels reduced both body ice content and in vivo erythrocyte injury. These results not only provided conclusive evidence for glucose's cryoprotective role in R. sylvatica, but also revealed that tissue glucose level is a critical determinant of freeze tolerance capacity in this species.

Acclimatization↗
PubMed1989

Preliminary study of water and some element contents in boar spermatozoa, before, during and after freezing.

Boar semen was analysed by electron microscopy coupled to image analysis and X-ray energy dispersive spectroscopy, during the usual process for freezing and thawing in field conditions. Freeze-substitution and freeze-quenching permitted recording of real or potential intracellular ice before, during, and after freezing. Heads and flagella displayed two different osmotic properties before freezing. Heads were dehydrated progressively before and during freezing, while flagella were hydrated before freezing and were only dehydrated during freezing. All parts of the thawed cells were rehydrated. Ice crystal damage was mostly present in frozen mitochondria and axonemes and the acrosomes were strongly affected by thawing. The total amounts of Na, Cl, Ca, K, Mg, and Zn per cell were only elevated in frozen and thawed midpieces while the heads were permeable both to water and elements at that time.

Animals↗
PubMed1990

Freeze-thaw-induced changes of the zona pellucida explains decreased rates of fertilization in frozen-thawed mouse oocytes.

Frozen-thawed oocytes have a reduced rate of fertilization (48.8%) when compared with unfrozen controls (97%). In this study we have used zona-drilling to bypass the zona pellucida and investigate whether the decreased rate of fertilization is due to freezing-induced changes in the zona pellucida which prevent sperm penetration. After zona drilling the fertilization rate of frozen-thawed oocytes (87.8%) was the same as for zona-intact unfrozen controls (88%), indicating that freeze-thaw-induced changes at the level of the zona pellucida were responsible for the decreased rate of fertilization. To determine whether the changes were occurring during the manipulations before and after freezing or the complete freeze-thaw cycle, oocytes were exposed to the complete set of manipulations normally experienced during cryopreservation and appropriate control groups. A small but significant decrease in the rate of fertilization (82.8%) was apparent in oocytes exposed to the manipulations before and after freezing compared with controls (92.2%). The freeze-thaw-induced changes in the zona pellucida therefore occur primarily during the complete freeze-thaw cycle itself and not the manipulations before and after freezing and are responsible for the decreased rate of fertilization observed in frozen-thawed oocytes.

Animals↗
PubMed1997

Effect of freezing on somatic cell count of quarter milk samples as determined by a Fossomatic electronic cell counter.

The objective of this study was to determine the effect of freezing and the duration of freezing on somatic cell count (SCC) in milk samples from quarters with no signs of clinical mastitis. Effect was determined by a fluoro-opto-electronic cell counter. Samples were frozen in a commercial freezer at approximately -20 degrees C. The milk samples were thawed at 1, 2, 3, 4, 7, 14, or 28 d. Another batch of samples was frozen 1 d, thawed, frozen again for 1 d, and then thawed. The SCC was determined on the fresh samples and after any one of the freezing periods. On average, the SCC were lower after all freezing periods than they were before freezing. However, the difference in SCC before and after freezing was small and relatively constant on the natural logarithm scale. The decrease in SCC was larger with longer freezing periods. When SCC thresholds of 200,000, 250,000, and 500,000 cells/ml were used to predict infection, freezing appeared to have little impact on the sensitivity and specificity of diagnostic parameters. Therefore, SCC, as determined by a fluoro-opto-electronic cell counter, determined for frozen quarter milk samples can be used in mastitis control and research programs.

Animals↗
PubMed1998

Bacterial cross-contamination of meat during liquid nitrogen immersion freezing.

Prerigor beef carcass surface tissue (BCT) was used to simulate lamb carcasses on a processing line with a 15-min liquid nitrogen (LN) immersion freezing step, and the potential for the dissemination of bacteria during freezing was examined. Streptomycin-resistant strains of Listeria innocua and Escherichia coli O157:H7 spiked into a fecal slurry were inoculated onto BCT pieces that were introduced into the freezing process to represent contaminated carcasses. Following this introduction, subsequently frozen uninoculated BCT, LN, and LN containers were examined for the inoculated organisms. In the first study, BCT samples were inoculated with ca. 7 log CFU/cm2 of both L. innocua and E. coli O157:H7, spray washed with water and frozen, distributed among uninoculated BCT, in LN for 15 min. In two separate trials, L. innocua was recovered by enrichment from all uninoculated BCT and LN samples. E. coli O157:H7 was also recovered from uninoculated BCT and LN, but this cross-contamination was more sporadic. Both species were recovered from the LN container following freezing. Attempts to enumerate cross-contaminating bacteria in the second trial indicated that contaminating levels were low (< 1.0 CFU/cm2 BCT). In a second study, a 2.0% lactic acid spray wash was used to reduce further the numbers of L. innocua introduced into the freezing system and resulted in fewer positive samples, although this organism was still recovered from many uninoculated BCT samples. When either bacterium was inoculated at lower initial levels (1.35 to 1.77 log CFU/cm2) and BCT was water or 2.0% lactic acid spray washed prior to freezing, neither L. innocua nor E. coli O157:H7 was recoverable by enrichment from uninoculated BCT, LN, or from the freezing container. Results demonstrate that bacterial cross-contamination of meat during LN immersion freezing can occur but indicate that the use of good sanitation practices and product with low microbial numbers can limit this occurrence.

Abattoirs↗
PubMed2001

Viability, efficacy, and storage stability of freeze-dried biocontrol agent Candida sake using different protective and rehydration media.

Viability, efficacy against Penicillium expansum on Golden Delicious apples, and storage stability of freeze-dried Candida sake strain CPA-1 were studied. The effect of several protective agents and rehydration media was investigated in the freeze drying of C. sake. Skimmed milk at 10% concentration was a good rehydration medium for all protectants tested. In general, good viability results were obtained when the same solution was used as a protectant and as a rehydration medium. The best survival was obtained when C. sake cells were protected with 10% lactose + 10% skimmed milk and rehydrated with skimmed milk (85% viability). The potential for biocontrol of the best freeze-dried treatments against P. expansum on apples was compared with that of fresh cells. Freeze-dried treatments at 1 x 10(7) CFU/ml reduced the incidence of decay by 45 to 66%. The best biocontrol effect was obtained with cells that had been freeze dried using 10% lactose + 10% skimmed milk as a protectant and 1% peptone as a rehydration medium, with a 66% reduction in rot incidence. However, in all treatments, the efficacy of freeze-dried cells was significantly lower than fresh cells. The stability of freeze-dried samples decreased during storage and was influenced by storage temperature. In the best treatment, storage of C. sake cells for 60 days at 4 degrees C resulte in final concentrations of 2.5 x 10(8) CFU/ml, which was a 10-fold reduction in relation to the initial starting concentration of cells prior to freeze drying.

Candida↗
PubMedDate not supplied

Evaluation of human sperm function after repeated freezing and thawing.

Sperm storage via freezing has been useful for men who have difficulty masturbating during assisted reproductive technology (ART) programs and before impotency caused by chemotherapy, vasectomy, and other procedures. Studies were undertaken to evaluate the extent of cryoinjury to sperm after repeated freezing and thawing. The results showed that normozoospermic and oligozoospermic sperm survived after 3 repeated freeze-thaw cycles. The inclusion of seminal plasma did not seem to protect human sperm during freezing and thawing. There were no significant differences in recovery percentages for motile, vital, and morphologically normal sperm between slow and rapid freezing methods in thaws 1, 2, and 3 of normozoospermic and oligozoospermic unwashed (u), washed (w), and washed + seminal plasma (ws) samples. However, there were significant percentage drops in the recovery of motile and vital sperm between each thaw (ie, first to second thaw, and second to third thaw) using both slow and rapid freezing for u, w, and ws samples (P < .01). There were also no significant differences in percentage recovery of motile, vital, and morphologically normal sperm between u, w, and ws samples during thaws 1 to 3 in the normozoospermic and oligozoospermic groups. Sperm were capable of fertilizing hamster oocytes microinjected with single sperms after 3 freeze-thaw cycles as evidenced by the formation of 2 distinct pronuclei and 2 polar bodies in 22.2% and 17.2% of normozoospermic and oligozoospermic samples, respectively. The numbers of normal vital motile sperm after 3 serial freeze-thaw cycles are adequate for bringing about fertilization via intracytoplasmic sperm injection in ART programs. Thus, leftover washed sperm in laboratories that perform in vitro fertilization can be frozen, thawed, and refrozen several times without loss of the sperms' ability to fertilize. This approach has tremendous benefits for men who have difficulty producing sperm and for those with low and declining sperm counts.

Animals↗
PubMed1992

Effect of freezing and thawing of serum on the immunoassay of lipoprotein(a).

We studied the effect of freezing and thawing of serum on the determination of lipoprotein(a) [Lp(a)] with a commercial enzyme-linked immunosorbent assay (ELISA) and an immunoturbidimetric assay (ITA). Portions of sera from 11 apparently healthy persons and pooled sera, from an additional 10 subjects were frozen at either -20 or -70 degrees C and thawed at room temperature. Cycles of freezing and thawing were repeated during the experiments (1 month). Samples were assayed for Lp(a) after thawing. Pooled sera were subjected to quick freezing at -70 degrees C and thawing at room temperature in cycles. Results show a significant (P less than 0.05) decrease in Lp(a) concentration in sera subjected to freezing and thawing. Samples thawed from -20 degrees C gave concentrations by ELISA that were significantly lower than those of fresh samples after one freeze-thaw cycle. By ITA the decrease was significant only after two cycles. In specimens frozen at -70 degrees C, Lp(a) concentrations determined by ELISA decreased after two cycles, and by ITA after three freeze-thaw cycles. Serum samples subjected to quick freezing at -70 degrees C and thawing did not show significant decreases in Lp(a) immunoreactivity during four cycles. Immunoreactivity of Lp(a) in samples stored at 4 degrees C decreased after 6 days but fell faster in serum samples subjected to freezing and thawing before storage at 4 degrees C.

Adult↗
PubMed2005

[The effect of freezing and low molecular weight heparin treatment on the production of hepatocyte growth factor of human RPE cells].

OBJECTIVE: This study is to investigate the human hepatocyte growth factor (HGF) level and proliferation of cultured human retinal pigment epithelial cells (RPE), treated with freezing, or Tedelparin (low molecular weight heparin), or both. METHOD: Cultured human RPE cells were placed at -80 degrees C freezer for 15 and 60 seconds respectively. One group of freezing-treated cells was kept in culture for another 3 d or 6 d, with or without Tedelparin (25 microl/ml). Another group of freezing-treated cells was injected into the vitreous of rabbits immediately, and 3 d or 6 d later the vitreous of injected rabbits were harvested and put into the medium of cultured human RPE cells, with or without Tedelparin (25 microl/ml). The level of HGF in culture medium and vitreous was measured by ELISA, and the cell proliferation was examined by MTT assay. RESULTS: The HGF concentration was increased on day 3 in culture medium of freezing-treated human RPE cells (F = 27.36, P < 0.01), and in the rabbit vitreous injected with freezing-treated human RPE cells (F = 6.24, P < 0.01). Tedelparin can reduce the HGF concentration in culture medium of freezing-treated human RPE cells at day 3 and 6 (F = 179.50, P < 0.01). The rabbit vitreous injected with freezing-treated RPE cells can promote the proliferation of normal RPE cells (P < 0.01), which was suppressed by 48 h treatment of Tedelparin (F = 44.90, P < 0.05). CONCLUSION: Freezing may increase the expression of HGF, or promote the proliferation of RPE cells. Tedelparin may suppress the proliferation of RPE cells by inhibiting HGF production.

Animals↗
PubMed2005

[Effects of cold-hardening on freezing tolerance and antioxidant enzyme activities in plantlets of Saussurea laniceps Hand.-Mazz].

Living conditions for plants in the mountains become increasingly less favorable with increasing altitude. In the alpine region, the plants are commonly exposed to daily rather than seasonal temperature fluctuations and by frequent freezing temperature. To elucidate the freezing tolerance mechanism of alpine plants, Saussurea laniceps Hand.-Mazz. was used as a model plant. It is a perennial herbal plant distributed in alpine regions of Yunnan and Tibet of China. It can survive on mountains with elevations over 4000 m. Wild S. laniceps plants are propagated only by seeds in the alpine areas. Micropropagation of S. laniceps through seed was a desirable method to get enough seedlings for freezing research. Micropropagation through plantlets derived from germinated S. laniceps seeds collected from Tibet was achieved successfully. Activities of antioxidant enzyme and solute contents were investigated in plantlets of S. laniceps. Freezing tolerance in plantlets increased after 7 or 15 d of cold-hardening (Table 1). Cold-hardening (2 degrees C) increased the activities of SOD, peroxidase, and catalase (Fig.1) in plantlets. A similar increase was also observed in the protein and proline content (Fig.1), whereas soluble carbohydrates changed little (Fig.1). These results obtained suggest that the higher activities of SOD, peroxidase, and catalase, as well as the higher protein and proline content may be biochemical adaptation for freezing toleranc in cold-hardened S. laniceps plantlets. Interestingly, deacclimation was slow; even after the plants were placed again under a temperature of 21-23 degrees C for 5 d, the higher freezing hardiness, enzyme activities, protein and proline content acquired after cold acclimation remained. In conclusion, our plantlet cultures have proved to be good materials for experimentation on freezing resistance in study of freezing-resistance mechanism in the alpine plant S. laniceps.

Adaptation, Physiological↗
PubMed1988

Effects of freezing and thawing on the viability and the ultrastructure of in vivo grown mycobacteria.

The influence of different frequencies of freezing-thawing cycles on the viability of in vivo grown mycobacteria was investigated. Pieces of armadillo tissues naturally or experimentally infected with Mycobacterium leprae were analyzed. The viability of M. leprae was determined by mouse foot pad titration. The viability of cultivable mycobacteria, sometimes present in armadillo tissues, was determined by culture. Electron-microscopic studies were performed on fresh or frozen-thawed armadillo tissues with natural leprosy and on livers of C57BL/6 mice experimentally infected with M. avium or M. lepraemurium. We found that the percentage of viable M. leprae bacilli is identical for naturally infected and experimentally infected tissues, frozen and thawed once. When the tissues were subjected to a second freezing-thawing cycle, a considerable loss of viability was observed (65%-97%). A third freezing-thawing cycle was lethal for most of the M. leprae cells, and after four freezing-thawing cycles no viable bacilli were found. The cultivable mycobacteria present in some armadillo tissues were found to be more resistant than M. leprae to freezing-thawing since these mycobacteria could still be cultivated after four freezing-thawing cycles. The results of the electron-microscopy study support the conclusion that M. leprae is more sensitive to freezing-thawing than the cultivable mycobacteria and show that the cytoplasmic membrane appears to be the target for the lethal action of freezing-thawing on mycobacterial cells. These results emphasize the importance of avoiding repeated thawing and refreezing of M. leprae-infected tissues when viable M. leprae cells need to be studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Freeze-dried segmental fibular allografts in azathioprine-treated dogs.

The successful use of a bone allograft may be negated by the host's immune response. This investigation assessed the efficacy of combining freeze-dried cortical allografts in three to six weeks azathioprine-immunosuppressed dogs. Forty-eight of 94 adult mongrel dogs were initiated for this study, and 46 of 94 were previously published and recompiled. The dogs were divided into five groups and followed for six months: Group I consisted of bilateral fresh autografts as an external control; Group II assessed the effect of freeze-drying on autogenous bone; Group III compared fresh autografts with fresh allografts; Group IV assessed the effect of freeze-drying on allografts; and Group V assessed the combined effect of placing freeze-dried allografts in immunosuppressed hosts. Biweekly roentgenograms were made to evaluate the time to union and the incidence of graft fatigue failure. Mechanical graft strength was assessed by rapid torsional loading to failure at the time of sacrifice. Biologic repair was assessed with the use of tetracycline and microradiographic techniques. The incorporation and repair of a fresh cortical autograft is better than that of a freeze-dried autograft because of fractures, nonunion, or delayed union of graft-host junctions; freeze-dried autografts have increased peripheral and internal resorption, yet an increased peripheral bony callus maintains normal graft strength; freeze-dried and fresh allografts are similar in roentgenographic characteristics, mechanical strength, and in the mechanism of graft incorporation; the use of three or six weeks azathioprine therapy did not improve the fate of freeze-dried allografts.

Animals↗
PubMed1978

Freeze-dried allogeneic segmental cortical-bone grafts in dogs.

Forty-four adult male mongrel dogs were used to compare segmental cortical freeze-dried allogeneic bone grafts with fresh autogenous, freeze-dried autogenous, and fresh allogeneic segmental cortical grafts. Group I consisted of bilateral fresh autografts as external controls; Group II, a fresh autograft on one side for internal control and a freeze-dried autogenous graft on the other side to evaluate the effect of freeze-drying on repair; Group III, a fresh autograft on one side and a fresh allograft on the other side to measure the differences between autogenous and allogeneic grafts; and Group IV, a fresh autograft on one side and a freeze-dried allogeneic graft on the other to see if freeze-drying altered the repair of allogeneic grafts. The grafts were analyzed qualitatively over a six-month period by the use of interval roentgenograms to determine the times of graft-host union and the incidence of fatigue fractures. Six months after operation, the repair processes in the four groups were compared quantitatively with respect to biological repair and physical strength using torsional stress-testing, tetracycline labeling, and microradiography. The results indicate both qualitatively and quantitatively that: (1) fresh bilateral segmental cortical autografts show reproducible characteristics, so that the canine fibula furnishes a satisfactory model (Group I); (2) freeze-drying does not inhibit the repair process per se (Group II); (3) fresh allografts are rejected in varying degrees of intensity (Group III); and (4) freeze-drying does not protect cortical allogeneic grafts from such rejection (Group IV).

Animals↗
PubMed2001

Freeze tolerance in the gray treefrog: cryoprotectant mobilization and organ dehydration.

Freeze tolerance in the frog Rana sylvatica is supported by nonanticipatory mobilization of cryoprotectant (glucose) and redistribution of organ water. Other freeze-tolerant frogs may manifest these responses but differences exist. For example, the gray treefrog (Hyla versicolor) accumulates mostly glycerol as opposed to glucose. The current study reports additional novel features about cryoprotection in H. versicolor. Frogs were acclimated to low temperature for 12 weeks and frozen for 3 days at -2.4 degrees C. Some frogs were then thawed at 3 degrees C for 4 hr. Calorimetry revealed that frozen frogs had 53.9% +/- 11.1% of their body water in ice, and all frogs recovered following this procedure. Plasma glucose was low prior to the onset of freezing (1.1 +/- 0.9 micromol/ml) and it was 20x higher in postfreeze frogs. Constituting nearly 30% of plasma solute, glycerol was 117.2 +/- 13.6 micromol/ml prior to freezing and it remained equally high in postfreeze frogs. Liver water content was moderately lower in frozen frogs when compared to controls (62.9% +/- 3.7% vs. 68.6% +/- 1.7%), whereas postfreeze frogs excessively hydrated their livers (75.7% +/- 2.1%). Less-pronounced changes were seen in muscle water content. H. versicolor can mobilize its major cryoprotectant, glycerol, in response to extended cold acclimation, which is unique in comparison to other freeze-tolerant frogs, and it experiences only moderate organ dehydration during freezing. This species conforms with other freeze-tolerant frogs, however, by mobilizing glucose as a direct response to tissue freezing.

Acclimatization↗
PubMed2000

Protein denaturation during freezing and thawing in phosphate buffer systems: monomeric and tetrameric beta-galactosidase.

During freezing in sodium and potassium phosphate (NaP and KP) buffer solutions, changes in pH may impact the stability of proteins. Since the degradation pathways for the model proteins, monomeric and tetrameric beta-galactosidase (beta-gal), chosen for this study are governed by conformational changes (i.e., physical instability) as opposed to chemical transformations, we explored how the stresses of freezing and thawing alter the protein's native structure and if preservation of the native conformation during freeze-thawing is a requisite for optimal recovery of activity. During freezing in NaP buffer, a significant pH decrease from 7.0 to as low as 3.8 was observed due to the selective precipitation of the disodium phosphate; however, the pH during freezing in KP buffer only increased by at most 0.3 pH units. pH-induced inactivation was evident as seen by the lower recovery of activity when freeze-thawing in NaP buffer as compared to KP buffer for both sources of beta-gal. In addition, we investigated the effects of cooling rate and warming rate on the recovery of activity for monomeric and tetrameric beta-gal. Optimal recovery of activity for the NaP samples was obtained when the processing protocol involved a fast cool/fast warm combination, which minimizes exposure to acidic conditions and concentrated solutes. Alterations in the native secondary structure of monomeric beta-gal as measured by infrared spectroscopy were more significant when freezing and thawing in NaP buffer as opposed to KP buffer. Conformational and activity analyses indicate that pH changes during freezing in NaP buffer contribute to denaturation of beta-gal. These results suggest that proteins formulated in NaP buffer should be frozen and thawed rapidly to minimize exposure to low pH and high buffer salts.

Buffers↗