Physical and Chemical Properties of Gels Application to Protein Nucleation Control in the Gel Acupuncture Technique

Journal of Crystal Growth 205 (1999) 375}381 Physical and chemical properties of gels Application to protein nucleation control in the gel acupuncture technique Abel Moreno! *, Gabriela JuaH rez-MartmH nez!, TomaH s HernaH ndez-PeH rez", Nikola Batina#, Manuel Mundo$, Alexander McPherson% !Departamento de Bioqun&mica, Instituto de Qun&mica, U.N.A.M. Circuito Exterior, C.U. Me&xico, D.F. 04510, Mexico "Departamento de Ciencias BaH sicas, Universidad Auto& noma Metropolitana, Azcapotzalco, Mexico #Departamento de Qun&mica, Universidad Auto& noma, Metropolitana-Iztapalapa, Mexico $Department of Nematology, University of California, Riverside, USA %Department of Biochemistry and Molecular Biology, University of California, Irvine, USA Received 1 May 1998; accepted 24 April 1999 Communicated by N.E. Chayen Abstract In this work, we present a new approach using analytical and optical techniques in order to determine the physical and chemical properties of silica gel, as well as the measurement of the pore size in the network of the gel by scanning electron microscopy. The gel acupuncture technique developed by GarcmHa-Ruiz et al. (Mater. Res. Bull 28 (1993) 541) GarcmH a-Ruiz and Moreno (Acta Crystallogr. D 50 (1994) 484) was used throughout the history of crystal growth. Several experiments were done in order to evaluate the nucleation control of model proteins (thaumatin I from Thaumatococcus daniellii, lysozyme from hen egg white and catalase from bovine liver) by the porous network of the gel. Finally, it is shown how the number and the size of the crystals obtained inside X-ray capillaries is controlled by the size of the porous structure of the gel. ( 1999 Elsevier Science B.V. All rights reserved. PACS: 36.20; 87.15; 81.10 Keywords: Protein crystal growth; Silica gels; Nucleation control; Lysozyme; Thaumatin I; Gel acupuncture technique 1. Introduction several "elds of materials science. In the case of biological macromolecules, most of the investiga- Nowadays, the availability of high-quality single tions have been focused on determining the best crystals for 3D X-ray characterisation is crucial in crystallisation conditions for a speci"c system. Al- though there have been some approaches to over- * Corresponding author. Tel.: #52-5-6224568; fax: #52-5- come this handicap, the next di$cult part of the 6162217. research needs to be focused on choosing the best E-mail address: [email protected] (A. Moreno) crystal growth technique in order to obtain huge 0022-0248/99/$ - see front matter ( 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 0 2 4 8 ( 9 9 ) 0 0 2 6 3 - 8 376 A. Moreno et al. / Journal of Crystal Growth 205 (1999) 375}381 single crystals. One interesting approach for ob- ing the gels is well established and all the para- taining high-quality single crystals is to reduce or at meters that control the gelling phenomena are well least minimise the convection phenomena. There understood. are three possible ways for doing this. The "rst is to grow crystals in low-gravity conditions. The second as shown by GarcmH a-Ruiz and Moreno [1,2], is to 2. Chemicals grow crystals inside thin capillary tubes just by keeping the unidirectional transport of the molecu- Thaumatin I (Sigma, T-7638), Lysozyme (Sigma, les through the true protein solution. The third is to L7773) and catalase (Sigma, C3155) were used as grow crystals inside the porous network though, in purchased without further puri"cation. Sodium po- the case of macromolecules, this seems a non- tassium tartrate, sodium chloride and ammonium recommended crystal growth method. The know- sulphate were used as precipitating agent solutions, ledge about the physical and chemical properties of respectively. All the solutions were prepared with gels is important for the gel acupuncture technique. double-distilled water. The layer that holds the This has been an alternative technique for growing capillaries was made of silica, by mixing appropri- crystals of biological macromolecules. For this par- ate volumes of sodium silicate solution (Aldrich, ticular case, the gel works as a porous network that 33,844-3) with a speci"c gravity of 1.06 g/cm and permits the transportation of the ions and keeps the acetic acid 1 M. The gels obtained by hydrolysis of capillaries well "xed in vertical position. These are tetramethoxysilane (TMOS, FLUKA catalogue the reasons for studying silica gels. The aim of this number 87682) were prepared by mixing the TMOS research will be focused on determining these phys- with double-distilled water, following the experi- ical and chemical properties of the silica gels ob- mental set-up described in the paragraph below. For tained for chemical synthesis as a polymeric evaluating the nucleation phenomena (using the im- reaction. There are some reports about the global plementation of the gel acupuncture technique) the porosity of gels and their fractal structure derived pH values of the silica gel were 8 and 10 for from tetraethoxysilane (TEOS), where microtexture thaumatin and 4.0 and 6.0 for lysozyme. We used has been studied [3]. Di!erential scanning phosphate bu!er (100 mM) to maintain the protein calorimetry has been used to measure the pore size solution at pH 7.0 for thaumatin and Tris-HCl pH distribution (PSDs) from the melting and freezing 9 (100 mM) for catalase. Sodium azide solution was curves of water con"ned in pores. Thermal po- added to the precipitating agent at 0.1% (w/v). rosimetry has been established by combining some physical properties applied to 12 kinds of commer- cial silica gels [4,5]. The determination of pore size 3. Experimental procedure distribution from sorption isotherms applying the percolation theory has been published by Zhdanov The gels used in this work were hydrogels. The et al. [6]. Silica membranes, their preparation and gels are two-component media with water molecu- the structure of microporous, have been published les soaking a porous #exible polymer network. The in Ref. [7]. The pore surface, characteristics of gelation process corresponds to the setting of macroporous silica-gels prepared from polymer a polymeric cluster stretching over the whole vol- containing solution, has been published by ume of solution. This process is either reversible for Nakanishi et al. [8]. There are many reports about physical gels, like gelatine and agarose gels, which the use of gels as crystal growth cells that take into are obtained by decreasing the temperature, or account their advantages for keeping the di!u- irreversible for chemical gels (such as silica or poly- sional path all over the experiment. Revision and acrilamide gels) which are obtained from the forma- data to prepare silica gels from di!erent ways have tion of strong bonds. Although universal `besta gel been published by Roberts et al. [9]. Many of the cannot be recommended [10], silica and agarose problems, found in the crystal growth of proteins, gels have proved their e$ciency for growing mac- could be overcome if the methodology for prepar- romolecular compounds [11,12]. A. Moreno et al. / Journal of Crystal Growth 205 (1999) 375}381 377 3.1. Silica gels obtained by neutralisation of sodium bution of porous "lled by the liquid phase. Due to metasilicate the small average pore size, the gel structure pre- vents convective mass #ow while allowing the The silica gels have been used for many years as Brownian motion of ions and small clusters crystal growth cells, because their porous network through the intraporous phase. The gelation time permits the di!usion of several ions and of some depends on many parameters: nature and concen- large polymers (like polyethyleneglycols and other tration of species in solution, pH, temperature. In macromolecules). These silica gels were basically case of thermally stable solutions, one can shorten obtained from the neutralisation of sodium meta- this time by increasing temperature. Practical con- silicate solution (1.06 g/ml) with acetic acid 1 M. siderations require gelation times to take place in Sodium metasilicate solution 1.06 g/ml was pre- less than a couple of days. One estimates that the pared by the following equation: gel is set when it resists pouring. It must stick to the < (0.06) crystal growth cells walls. It can look somewhat < " ! opalescent but without heterogeneities such as "s- !o3! 1.39 sures. where <11 is the volume of sodium silicate solution There are some useful ways to determine the to be taken from the bottle of the chemical (AL- gelation time for many type of gels. The "rst DRICH Cod. 33,844-3). <! is the volume to be method consists of measurements from the polym- measured in a volumetric #ask, 1.39 is the density of erisation process, the time when the gel structure is the sodium metasilicate from the chemical and well "xed to the walls of the container. This means o3! is the density of water at room temperature. the time when the gel does not pour anymore. It is Once the solution of sodium metasilicate has been obvious that the whole amount of water is encap- prepared, a titration plot is then obtained in order to sulated inside the porous network. As a result of have several gels at di!erent pH values for determin- this macromolecular inter-crossing from the poly- ing the gelling time and the reproducibility in the meric reaction, the structure of the network expels experiments. This could be a crucial parameter in the water drops by itself. The process of expelling drops application of these gels to protein crystallisation. of water is usually called syneresis, which is the second technique for estimating the gelation time. 3.2. Silica gels from tetramethoxysilane (TMOS) This is UV-VIS Spectroscopy using quartz cells and solutions measuring at 250 nm.

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