A Brief Analysis of Capillary Sealing Methods and Their Effectiveness Darren S. Ferree and Mark J. van der Woerd* Universities Space Research Association, NASA Laboratory for Structural Biology, Code SD46, NASA MSFC, Huntsville, Alabama 35812 Received November 1, 2002;
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Revised Manuscript Received December 12, 2002
ABSTRACT: Protein crystals and the solutions from which they are grown must be carefully protected against dehydration. In the absence of a proper seal, the crystals will dehydrate and lose their original properties and subsequently the ability to diffract X-rays. We conducted a systematic study of the efficacy of the most common sealing materials in use and we report the results here. The study shows that no single material is completely effective in both untreated and silanized glass capillaries. Various combinations of two materials, however, give excellent seals that will prevent dehydration for long periods of time. Recommendations are made for sealing materials to be used. Introduction Sealing protein crystals and crystallization solutions together in capillary tubes proved to be a critical enabling step in determining protein structures by X-ray diffraction studies.1 Protein crystals typically contain between 30 and 70% solvent.2 In the absence of this solvent, the crystals physically shrink, become disordered, and then lose the ability to efficiently diffract X-rays. It is therefore important to preserve the crystals in a hydrated state. One of the techniques used is storing the crystal and a small amount of its crystallization solution inside a sealed capillary tube to keep the crystal hydrated. Typically, capillaries have been sealed with materials including wax3, clay, or epoxy glue. Before the advent of cryocooling techniques (for a review see Garman et al.4), the universal technique to protect crystals during X-ray structural studies was capillary mounting. Glass capillaries provide several other advantages: although they absorb X-rays and also scatter them to contribute to the image background, they do not contribute to the X-ray diffraction pattern and they are optically clear so that the crystal position and orientation are immediately apparent. Capillary mounting continues to have a place in structural studies. Initial screening for crystal quality is still performed at room temperature in thin-walled capillaries. After determination of crystallization conditions, the next step is typically the cryopreservation of crystals. However, there are cases in which a suitable cryoprotectant cannot be found and capillaries are used for data collection. Neutron diffraction, a relatively new method, allows for the accurate determination of the position of hydrogen atoms in macromolecules. This technique was first used by Wlodawer et al. to show the position and function of the hydrogen atoms in RNase A.5 The crystals required for this technique are relatively large and radiation damage does not occur. Therefore, the preferred mounting technique is glass capillaries. The duration of experiments in neutron diffraction work, unlike X-ray diffraction work, is typically on the order * To whom correspondence should be addressed. Mark J. van der Woerd. Phone (256) 544-3343. FAX (256) 544-5543. E-mail
[email protected].
of weeks or months, rather than hours to days. Therefore, effective sealing methods are essential for neutron diffraction work. The growth of crystals inside glass capillaries requires similar long-term sealing techniques. The use of capillaries for crystal growth is an advantage for crystals that are sensitive to handling, for example, those with high solvent contents. Capillaries are also attractive because they take up very little space and normally provide easy and complete visual access to the crystal growth environment. These properties of capillaries are particularly important for use in an environment in which limited space or weight are available, as is the case for microgravity experiments. Koszelak et al.6 reported the first use of this efficient technique, but the material used was not glass and the sealing technique was accomplished by melting the container ends. Alvarado et al. reported the successful use of glass capillaries in microgravity for protein crystal growth of a human Bence-Jones protein.7 Unfortunately, they report that their capillaries were “capped”, and no further information is available for the sealing technique used. In addition, Lopez-Jaramillo et al. have successfully designed a new method of crystal growth inside glass capillaries, in which the crystals are never individually manipulated.8 Their method was recently used for the first time to determine a crystal structure without ever physically touching the crystals themselves.9 We have both transported and grown crystals in capillaries for neutron diffraction experiments in our recent work. Maintaining effective capillary seals has been of considerable interest to us. In this study, we report on a number of simple diagnostic experiments we performed that have allowed us to optimize our sealing methods and thereby maintain our crystal samples for as long as possible. Experimental Section Borosilicate glass capillary tubes (VitroCom, Inc., Mountain Lakes, NJ) with a 1.0 × 1.0 mm inner diameter square crosssection, 50 mm in length, and a 0.2 mm wall thickness were silanized by drawing a 2% Aquasil (Sigma Chemical Corporation, St. Louis, MO) solution through them with vacuum, then
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Figure 1. Mass change in silanized capillaries sealed with beeswax. Solid lines describe experimental data, while dashed lines describe controls, which were sealed but did not contain liquid. rinsing with an equal volume of water and allowing them to dry. Capillaries designated as untreated were used without any additional cleaning or treatment. Slugs of dyed water were drawn into the tubes by capillarity and then positioned in the center of the tube by using air pressure on its end. Heat-fused capillary tubes were sealed by applying the flame of a propane torch to the ends of the tubes until fused. Capillaries were sealed with beeswax (Hampton Research, Laguna Niguel, CA) by filling the ends of the tubes with molten beeswax and allowing them to cool. For sealing with Critoseal (Fisher Scientific, Suwanee, GA)sa product originally designed for hematological determinations in capillariessthe ends of capillaries were pressed into Critoseal and then withdrawn. Green mounting clay (Hampton Research) and high vacuum grease (Dow Chemical Corporation, Midland, MI) were used in the same manner as Critoseal. Epoxy glue (Cole-Parmer Instrument Company, Vernon Hills, IL) was used to fill the ends of the capillaries and was then allowed to cure. Capillaries sealed with combinations of beeswax and epoxy, Critoseal and epoxy, or clay and epoxy were first sealed at the ends with beeswax, Critoseal, or clay and then dipped in epoxy and allowed to cure. Each seal efficacy determination was performed in sets of a minimum of five capillaries. Two types of controls were made, the first by sealing capillary tubes without dyed water, the second by leaving tubes with dyed water unsealed. The control experiments were executed in sets of two. Each capillary tube was weighed on an analytical balance (Mettler Toledo AG204 DeltaRange) immediately after filling and sealing, and was then weighed at intervals to establish trends over time. The capillaries were handled with sterile gloves and were stored in a desiccator cabinet at room temperature.
Results and Discussion The results for the capillary sealing tests can be divided into four different groups based on the tests performed: short duration in untreated capillaries, long duration in untreated capillaries, short duration in silanized capillaries, and long duration in silanized capillaries. We present these grouped results and discuss a comparison below. To establish the efficacy of the materials studied for long- and short-term capillary sealing, the weights of
capillaries filled with approximately equal amounts of dyed water were followed over time. An example of these data is displayed in Figure 1 for silanized capillaries sealed with beeswax, followed for 53 days. Data were collected for each sealing method using both silanized and untreated capillaries. As shown in Figure 1, for each method we used two control samples, which did not contain liquid. We followed the weight change of these samples over time to gain an understanding of possible weight change of the seal itself and to capture the trends in systematic errors. All the data points at a specific point in time were transferred to graphs as shown in Figure 2, described below. Short duration tests (28 days) for seal integrity in untreated capillaries were performed for six different sealing methods, one of which is the heat-sealed method, in which the capillaries are closed by melting the glass with a torch (Figure 2a, triangles). In Figure 2, each set of measurements is displayed with a pair of open and closed symbols, showing experimental data and controls, respectively. The empty heat-fused capillaries neither gain nor lose appreciable mass over the course of the experiment. The actual experiment with heatfused capillaries filled with liquid shows an average increase in mass of about 0.6 mg. These data points serve as our reference, because we presumed heatsealed glass to provide the ideal seal. Clay (Figure 2a, crosses) and epoxy glue (diamonds) show nearly perfect performance over this time span, with average weight losses of less than 1 mg. Critoseal (circles), a clay-like material which is forced into the ends of the capillaries, also seals well with weight losses observed typically less than 2 mg and only one exception for which more water evaporated. Seals made by melting beeswax and dipping the capillary ends into the wax are unreliable (squares), with observed weight loss of 10 mg or more. Finally, vacuum grease proved ineffective in sealing capillaries (asterisks in Figure 2a).
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Figure 2. (a) Mass change in untreated capillaries after 28 days. Symbols displayed in the legend describe the materials used. Open symbols describe the experimental values, while filled symbols describe the associated results for controls, which are sealed but do not contain liquid. The series are displayed left to right from best performing to worst performing seals, as determined by weight loss. (b) Mass change in silanized capillaries after 22 days. The definition of the symbols is the same as in panel a. Symbols are again ordered left to right from best performing to worst performing seals, as determined by weight loss.
Seal integrities in capillaries, which were silanized treated with Aquasil, a hydrophobic agent commonly used both to prevent adhesion of protein crystals to surfaces and to facilitate crystal mounting procedures, follow a somewhat different behavior over a 22-day period (Figure 2b). In Figure 2b, the same distinction has been made to describe experimental data and control data side-by-side by open and closed symbols, respectively. Sealing with heat (triangles) showed a variability of weight of approximately (2 mg, while the weight change for the controls was within (1 mg. Mounting clay (crosses) appeared to provide an excellent seal, but failed completely in one case. Epoxy glue (diamonds) tends to give good seals, but two out of 10 capillaries sealed that way lost considerable weight (4 and 17 mg). Critoseal (circles) performs well as a seal with only one instance of a 2 mg loss, which is similar to the case for heat treatment. Beeswax (squares) seals silanized capillaries reasonably well, with weight losses ranging from 0.5 to 4.5 mg. Vacuum grease (asterisks) performs unacceptably with weight losses from 5 to more than 20 mg. Figure 2b also includes data for evaporation from capillaries which were deliberately left unsealed to allow the evaluation of data in reference to two extremes: heat sealed glass and completely open glass. On the basis of the results detailed above, it was prudent to explore more reliable sealing methods compatible with the use of protein solutions. The following combinations of seals were tested: critoseal and epoxy, beeswax and epoxy, and clay and epoxy. These methods were chosen based on the results of the single material sealing experiment, keeping in mind that the use of a blowtorch is not compatible with the use of protein solutions and that the use of vacuum grease is clearly inferior to the other methods. Both untreated and silanized glass capillaries were periodically weighed up to 390 days from the start of
the experiment. For the untreated glass, the combinations clay and epoxy (crosses), beeswax and epoxy (squares), and Critoseal and epoxy (circles) were tested. The results are displayed in Figure 3a. Two of the three combinations, clay and epoxy, and beeswax and epoxy show virtually no weight loss (compared to control experiments described in Figure 2a,b). The third combination of sealing materials, Critoseal and epoxy, clearly shows considerable weight loss. Figure 3b shows the equivalent data for silanized capillaries, using symbols for each combination of sealing material as used in panel a. In this case, none of the three methods show appreciable weight loss over 390 days. Weighing capillaries on an analytical balance and recording any measurable change in weight provided the data we collected for these experiments. Weight determination was chosen as the quantitative metric for this experiment because the associated errors are exceedingly small compared to any small volume determination. The correct use of control capillaries allows for exclusion of the possibility that a sealing material dehydrates or attracts water, which events will be reflected in a weight change in the capillaries sealed without dyed water. Thus, the controls display part of the systematic errors involved in the method used. When the actual weight determinations are outside the range of the control measurements, these results are indicative of true evaporative losses and not due to change in sealing material. In a few apparent cases in our data, we observe an appreciable increase of mass, which cannot be reasonably explained. Reviewing the experimental procedures, in which the capillaries were all stored in one container, it is possible that a significant increase in weight may be caused by addition of a piece of extraneous matter, perhaps clay or grease from another experiment. Gener-
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Figure 3. (a) Mass change in untreated capillaries after 390 days. Symbols displayed in the legend describe the combination of sealing materials used. Open symbols describe the experimental values, while filled symbols describe the appropriate control values. (b) Mass change in silanized capillaries after 390 days. The definition of the symbols is the same as in panel a. Symbols are again ordered left to right from best performing to worst performing seals, as determined by weight loss.
ally, however, the data show clear trends: few, if any, single seal materials are reliable for sealing beyond a short time span. Sealing methods consisting of two materials almost always gave a reliable seal, with the exception of the Critoseal and epoxy combination in untreated capillaries. This last data point makes clear that in general the sealing efficacy for any compound depends on the material properties of both the sealing material and the surface to which it is applied. We have found that the following combinations work well for sealing untreated glass: clay and epoxy, and beeswax and epoxy. For silanized glass surfaces we find that the combinations of clay and epoxy, beeswax and epoxy, and Critoseal and epoxy work adequately. The capillaries used in this study were not representative of those used for diffraction experiments, but the results presented here hold true for any capillary that does not show significant evaporative loss due to wall porosity. Capillaries for diffraction data collection have very thin walls, typically 0.01 mm, and become more fragile as the internal diameter increases. Sealing procedures that do not require pressing the capillary into a material are therefore preferred. The combination of beeswax and epoxy appears to be the most universally acceptable method for sealing thin-walled capillaries, both silanized and untreated. Conclusions The data presented clearly demonstrate that for longterm and short-term applications requiring seals to prevent evaporative loss from capillaries some single materials seal adequately. In both silanized and untreated glass capillaries acceptable materials for shortduration sealing are clay, epoxy, or Critoseal. For long-
duration sealingsand implicitly for a better quality seal during short-duration sealingsthe data show that a combination of sealing materials is required. Both for untreated glass and for silanized glass the combination of beeswax followed by an application of epoxy glue has proven highly effectual. This combination is also compatible with the use of thin-walled capillaries as used for X-ray diffraction analysis of protein crystals. Acknowledgment. The authors wish to acknowledge Drs. Lisa Monaco and Edward Snell for discussing this work and for providing critical input to the manuscript. This work was done as part of NASA’s Iterative Biological Crystallization project. References (1) Bernal, J. D.; Crowfoot, D. Nature (London) 1934, 133, 794795. (2) Matthews, B. W. J. Mol. Biol. 1968, 33, 491. (3) Rayment, I. In Treatment and Manipulation of Crystals; Methods in Enzymology; Wyckoff, H. W., Hirs, C. H. W., Timasheff, S. N. Eds.; Academic Press: Orlando, FL, 1985; Vol. 114A, pp 136-140. (4) Garman, E. F.; Schneider, T. R. J. Appl. Crystallogr. 1997, 30, 211-237. (5) Wlodawer, A.; Sjolin, L. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 1418-1422. (6) Koszelak, S.; Leja, C.; McPherson, A. Biotechnol. Bioeng. 1996, 52, 449-458. (7) Alvarado, U. R.; DeWitt, C. R.; Shultz, B. B.; Ramsland, P. A.; Edmundson, A. B. J. Cryst. Growth 2001, 223, 407-414. (8) Lopez-Jaramillo, F. J.; Garcia-Ruiz, J. M.; Gavira, J. A.; Otalora, F. J. Appl. Crystallogr. 2001, 34, 365-370. (9) Gavira, J. A.; Toh, D.; Lopez-Jaramillo, J.; Garcia-Ruiz, J. M.; Ng, J. D. Acta Crystallogr., Section D 2002, 58, 1147-1154.
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