EFFECT OF “FREEZING” TREATMENTS ON THE HYDROTHERMAL STABILITY OF COLLAGEN
STEPHEN L. WILLIAMS, SARAH R. BEYER, & SAMINA KHAN
ABSTRACT—In museum settings, subjecting objects to temperatures below 0°C has become an acceptable treatment for controlling pests, stabilizing water-saturated materials, and preserving organismal materials. Questions regarding the effect of “freezing” on collagen stability prompted an investigation of the temperatures at which collagen shrinks using microscopic analysis. Results indicate that both desiccation and freezing of mammal skin tissue tend to reduce shrinkage temperature, and that freezing for pest control and stabilization of water-saturated skin material has minimal initial effect on shrinkage temperature. Fresh skin material that is frozen, thawed, and dried showed significantly lower shrinkage temperatures, compared to other groups tested.
Subjecting objects and specimens to temperatures below 0°C has become a common conservation treatment in museum settings for pest control (Florian 1990a; Strang 1992), stabilization of water-saturated materials (Jakes and Mitchell 1992; Koesterer and Geating 1976), temporary preservation of unprepared biological materials (Dickerman and Villa-R. 1964; McConachie 1993), and long-term preservation through freeze-drying procedures (Hower 1970). However, the implications of this process, often referred to as “freezing,” are misleading because water molecules may or may not be in a solid state, depending on the materials and temperatures involved (Florian 1990b). In this paper, “freezing” refers to the process of subjecting materials to temperatures below 0°C, not to the inducing of a phase transition of water molecules.
Although various freezing treatments have been widely and successfully used in museums, it seems that the decision to use this treatment is based on theory or on information extrapolated from a few obscure sources and often does not take its effects into account. Florian (1990b) provides a good review of the current state of knowledge about freezing museum materials. For some materials, however, there is a need for basic, systematic study of material reaction to various freezing treatments.
This need became clear in a recent emerency in which dried skin material had become water-saturated and required treatment. Would freezing treatments for wood, paper, textiles, and even leather be applicable for water-saturated skin material that was normally preserved in a dry condition? The current study analyzes the temperature at which collagen shrinks to gain a better understanding of how fresh and dried skin material responds to freezing treatments. The information gained is relevant to supporting or refuting the wisdom of current freezing practices in museum settings.
Collagen is the most common protein in mammal skin and leather. Thus treatments for its preservation are relevant to collections of history, clothing and textiles, ethnography, and natural sciences as well as libraries. The collagen molecule consists of three left-handed helices twisted together in a right-handed direction. These helices are linked by hydrogen bonding. Under normal conditions high temperatures (65–75°C) are required to break these bonds and cause the collagen to collapse or shrink (Haines 1987). However, the shrinkage temperature (Ts) may be reduced, indicating a loss of collagen stability, by degradation processes and by some preservation treatments. Monitoring Ts change resulting from different treatments provides a mechanism for evaluation (Young and Grimstad 1990; Williams 1991; Larsen et al. 1993).
2 METHOD AND MATERIALS
This study was conducted in April and May 1994 at the Natural Science Research Laboratory of the Museum of Texas Tech University. During the study, ambient temperature and relative humidity were approximately 23°C and 35%, respectively.
Variables affecting results of Ts analyses include species, age, sampling location on skin, treatment, and agents of deterioration (Hobbs 1940; Gustavson 1956; Williams 1991). To focus on the relationships of various treatments, it was essential to know the natural condition of skin, have a fully documented history of treatments, and minimize influential variables. Thus a fresh skin from a single, adult, female, nonlaboratory rodent (Spermophilus tridecemlineatus; voucher reference number, SLW 7204; head and body length, 162 mm) was selected for study.
The middorsal region of the pelt was subdivided into six sectors, five of which were subjected to various desiccation and/or freezing treatments, and one which served as the control. Desiccation was performed in the dark under ambient conditions with the pelt pinned to an Ethafoam block. The time allowed for drying far exceeded limits specified by Michalski (1992). Freezing of encapsulated skin tissue was performed in a standard freezer at −8°C. The results of six different treatments are summarized in table 1. Shrinkage temperature was documented using a microscope (100 × magnification) with a central processor (Mettler FP90), hot-stage (Mettler FP82HT), and a recording printer (Epson FX870). Sample preparations involved presoaking in distilled water and using described methods (Young 1987; Young and Grimstad 1990; Williams 1991). Temperature was increased 2.0°C per minute. Temperature for initial and final shrinkage stages were documented 10 times for each of the 6 groups. Because movement of collagen fibrils may be caused by extraneous factors (Williams 1991), the initial Ts was recorded when simultaneous movement of two or more fibrils was observed in the field of vision. The final Ts was recorded when all movement in the field of vision stopped for at least 10 seconds. Because Ts is properly regarded as a temperature range between the initial and final stages (Gustavson 1956), the midpoint of the range has been used for designating specific temperature values (Von Hippel and Wong 1963); this method facilitated reporting of results in the current study.
TABLE 1 SUMMARY OF TREATMENTS APPLIED TO FRESH SKIN TISSUE
The SPSS program ONEWAY (Norvsis/ SPSS Inc. 1990) was used to obtain standard statistics for the 10 Ts analyses of each group, test homogeneity of variances, and perform analysis of variance between groups. The Student-Newman-Keul procedure of the same program was used to identify nonsignificant subsets between groups.
3 RESULTS AND DISCUSSION
Using the midpoint between initial Ts and final Ts, differences in Ts were noted between treatment groups (table 2). The Fmax-test for homogeneity of variances (Fmax = 3.807) showed no significant differences (P ≥ 0.05) among groups. The analysis of variance showed Ts differs significantly (F = 30.19) between groups. The Student-Newman-Keul procedure identified five nonsignificant subsets, with groups 1 and 4 each being significantly different from all other groups (table 2).
TABLE 2 SHRINKAGE TEMPERATURE ANALYSES (GROUPS FROM TABLE 1 LISTED IN DECREASING ORDER BY MEAN VALUE)
The most stable condition for collagen was provided by the untreated control group, which had a mean Ts of 71.3. This higher than expected value (see Haines 1987) may demon-strate a need to study Ts of skin tissues of nonconventional species.
The fact that the mean Ts of group 1 (control) is significantly greater than that in the other groups indicates that treatments involving desiccation or freezing generally contribute to collagen destabilization. A comparison of the group means shows, however, that groups desiccated first tend to be more stable than groups that were frozen first. This trend may be related to the denaturization of some proteins by phase transitions of water during freeze-thaw cycles, unfrozen water catalyzing hydrolytic or enzymatic activity, or radicals from lipid degradation (Matsumoto 1980; Florian 1990b).
Certain treatments are particularly relevant to museum practices. For instance, group 6 (dried and frozen) simulates current freezing practices for pest control (Florian 1990a). Although the mean Ts of group 6 is slightly lower than group 2 (dried only), it is reassuring to learn that the difference is not significant. These data suggest freezing dried skin tissues has minimal effect on collagen stability. Similarly, a comparison of group 2 (dried only) to group 5 (dried, rehydrated, and frozen), provides information regarding freeze-dry treatments of skin and leather that have become water-saturated, possibly as a result of disaster situations. Again, it is reassuring to find that freezing of water-saturated skin material had no significant effect on collagen stability. Although group 5 had a slightly higher mean Ts than group 2, the difference was not significant and could be explained by three Ts values of group 5 having higher than expected final temperatures.
A comparison of the Ts of groups 3 and 4 (frozen first) with the Ts of group 5 (dried, rehydrated, and frozen) is interesting because each group was frozen with unbound water present. The difference is significant. One explanation is that the phase transition of water in fresh skin involves intramolecular space, thus hydrogen bonding is negatively affected, as some unbound water may be expanded in a frozen state. Once the skin has been desiccated, it is possible that the addition of unbound water through rehydration processes involves primarily intermolecular spaces; thus the phase transition of water has little or no effect on hydrogen bonding. An alternative explanation is that water exists in the intramolecular spaces, and most is not frozen at the temperatures used in this study (Florian 1990b). If this is the case, however, collagen stability might be affected by other degradation processes.
The findings from group 4 (frozen, thawed, and dried) suggest a need for altering the common practice, particularly in vertebrate natural history collections, of freezing animals so that standard preservation techniques can be performed at a later time. The Ts for group 4 (mean = 66.1) was significantly lower than all other groups. The fact that the Ts was significantly lower than that of group 3 suggests that the freezing and subsequent desiccation had cumulative negative effects on collagen stability. Florian (1990b) also has recognized preservation problems that stem from freezing fresh skin tissues.
After visually inspecting skin or leather subjected to freezing treatment and noting little or no change, one might question why there would be a concern for a reduction in Ts. One concern might be the possible cumulative effects of treatment, especially treatment given to group 4. Perhaps the greater concern is that the loss of collagen stability potentially makes the material more susceptible to attack by proteolytic enzymes and possibly other mechanisms of deterioration (Stroz et al. 1993; Glew et al. 1994). Degraded skin material may also deteriorate faster than more stable forms. Young and Grimstad (1990) suggest that this possibility deserves further investigation. To fully appreciate the importance of the noted losses in Ts, it would be desirable to have a better understanding of the lower Ts limits provided by severely degraded collagen to evaluate the impact of treatments in terms of percent change to the potential degradation possible.
While freezing for pest control and for stabilizing water-saturated skin material may have minimal effect on collagen stability, it is important to realize that other components of the skin or leather, such as lipids, may not demonstrate the same level of stability (Florian 1990b). Furthermore, additional testing is needed to determine if repeated treatment, such as freezing for pest control, has any deleterious effects on the stability of collagenous tissues.
The current study, although simple in concept, provides potentially useful information regarding some standard museum practices. Results of this study indicate that current practices of lowering temperatures for pest control (Florian 1990a) and for stabilizing water-saturated materials (Jakes and Mitchell 1992) have minimal effect on collagen stability. Treatments involving the freezing, thawing, and drying of fresh skin tissue, however, contribute to collagen instability and are best avoided. All so-called freezing treatments of collagenous materials should be used cautiously. Not only may they fail to achieve the desired results, but they have the potential to promote reactions with other components of the skin and, through repeated treatments, to contribute cumulative effects about which little is known.
The authors gratefully acknowledge Gregory S. Young, Canadian Conservation Institute, for critically reviewing the manuscript; Michael R. Willig, Department of Biological Sciences, Texas Tech University, for assisting with statistical analyses; and the Leather Research Institute, Texas Tech University, for funding the project.
SOURCES OF MATERIALSCentral Processor (FP90) and hot-stage (FP82HT):
Mettler Instrument Corporation, P.O. Box 71, Hightstown, N.J. 08520–0071
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STEPHEN L. WILLIAMS is collections manager at the Natural Science Research Laboratory of the Museum of Texas Tech University and adjunct professor in the graduate Museum Science Program, Texas Tech University. He has a B.S. and an M.S. in zoology and an M.A. in museum sciences. Between 1976 and 1990, Williams was collection manager at the Carnegie Museum of Natural History. During this time he participated in the development of the Society for the Preservation of Natural History Collections, the Bay Foundation pilot training program in Los Angeles, and IMS conservation projects and activities. In 1990, Williams moved to Texas Tech University, where he manages the vertebrate research collections, publishes on collection management and care, and teaches preventive conservation. Address: Natural Science Research Laboratory, Museum of Texas Tech University, Box 43191, Lubbock, Tex. 79409–3191.
SARAH R. BEYER received a B.S. in textile design from the Philadelphia College of Textiles and Science. From 1985 to 1990, she worked as a textile designer for Burlington Industries. In 1992, she received an M.A. in museum sciences at Texas Tech University, and she is currently working on her doctorate in the School of Human Sciences at Texas Tech University. Address: College of Human Sciences, Texas Tech University, Box 41162, Lubbock, Tex. 79409–1162.
SAMINA KHAN is an associate professor in the Department of Merchandising, Environmental Design, and Consumer Economics in the School of Human Sciences, Texas Tech University. She received her M.S. from the University of Illinois and Ph.D. in textile science and clothing from Texas Women's University, Denton, Texas. She has been on the faculty at Texas Tech University since 1978. During this time she has conducted research and published on many aspects of natural textiles, including thermal, solar optical, and insulative properties as well as properties of detergency. She currently teaches textile conservation, principles of textile analysis, and historic costumes. Address: College of Human Sciences, Texas Tech University, Box 41162, Lubbock, Tex. 79409–1162.