Mechanistic and Structural Features of Protein Adsorption onto Mesoporous Silicates Joseph Deere, Edmond Magner,* J. Gerard Wall, and B. Kieran Hodnett Materials and Surface Science Institute and Department of Chemical and EnVironmental Sciences, UniVersity of Limerick, Limerick, Ireland ReceiVed: October 25, 2001; In Final Form: February 25, 2002 The adsorption of cytochrome c onto a range of different mesoporous silicates (MPS) was studied. The materials used, templated using both cationic and nonionic surfactants, have average pore-size diameters in the range from 28 to 130 Å. Cytochrome c was found to bind to all MPS investigated, with the pore diameter of the material, which was measured by N 2 gas adsorption, being crucial to mesopore penetration. The adsorption of a range of proteins with isoelectric points between 1 and 10 was investigated. For adsorption to occur, the surface charges of the protein and of the MPS must be complementary, in addition to the requirement that the pore diameter be sufficiently large. Pepsin at pH 6.5, for example, is negatively charged and does not adsorb onto cyano-modified silicate whereas subtilisin, which is of a similar size and bears an overall positive charge, is adsorbed. Using resonance Raman spectroscopy, cytochrome c was observed to occur in both high spin and low spin states, in contrast to that in solution, where the protein is predominantly in the low spin state. The presence of the high spin state may account for the enhanced peroxidative activity of the adsorbed protein. Introduction Mesoporous silicates (MPS) have been the subject of much interest since they were first described by Beck et al. in 1992 1 . MPS possess large surface areas (up to 1000 m 2 g -1 ), highly ordered pore structures and very tight pore size distributions (PSD); properties which have made these materials attractive candidates for a wide range of applications in catalysis, 2,3 sensor, 4,5 and separation technologies. 6,7 These materials have pore channels of diameter 1.5 to 10 nm which are of a similar size range to small proteins, and in particular, globular proteins. MPS possess a number of additional attributes, which make them attractive candidates for the immobilization of proteins. It is possible to chemically modify their surfaces with various functional groups, enabling electrostatic attraction or repulsion between an MPS and the biological molecule of interest to be maximized. 8 As a result of their silicate inorganic framework, MPS are chemically and mechanically stable and are resistant to microbial attack. Materials such as sol-gels display similar stability to MPS and have been used to encapsulate proteins for use as biosensors. 9,10 However, sol-gels suffer from the disadvantage of possessing a highly variable pore size distribu- tion (PSD). More importantly, their preparation can involve the use of harsh conditions or reagents, which are detrimental to, and can cause denaturation of proteins. 11 Using MPS, protein encapsulation occurs after synthesis of the support, avoiding this difficulty. MPS therefore hold great promise for use as supports to immobilize enzymes and may find applications in biosensors, 12 biocatalytic 13 and biomolecule separation systems. 8 Protein adsorption/immobilization onto silicate and other inorganic matrixes has been reviewed by Weetall 14 and numer- ous studies of protein adsorption onto silicate surfaces are to be found in the literature. 15-17 In the 1970s, Weetall et al. pioneered the use of porous inorganic materials for the im- mobilization of biological molecules and in particular the use of controlled pore glass (CPG). 18-23 CPG materials of pore sizes ranging from 300 to 2000 Å have frequently been reported in such studies, and generally, it has been found that the pore size of the CPG needs to be significantly larger than the biomolecule of interest. For instance, the activity of amyloglucosidase sharply decreased when the CPG pore size was less than 300 Å, and maximal activity was reported for a pore size of 400 Å. The enzyme loading was a direct consequence of both the pore size of the CPG and its surface area, with maximal activity occurring with material possessing both an optimal pore size and an optimal surface area. 19 The major disadvantages in using such materials are their cost and more importantly their surface area, which rapidly decreases with increasing pore size. 19,23 There have been a number of reports describing the use of MPS to immobilize proteins. 8,12,13,24-30 Balkus et al. 12,24 have immobilized cytochrome c (cyt c), papain, and trypsin onto MCM-41, SBA-15, and layered niobium oxide NB-TMS4. They have shown, as have Stucky et al.; 8 that the adsorption of proteins is dependent on the pore size of the material with, for example, adsorption of peroxidase onto MCM-41 being re- stricted due to the pore sizes being smaller than the enzyme. 24 Values of pH less than 7.0 were found to favor adsorption of papain and trypsin on MCM-41, whereas for cyt c, adsorption was most efficient at pH greater than 7.0. Desorption of papain occurred above pH 9.0, whereas no cyt c was desorbed at this pH. Cyt c immobilized onto MPS was stable under what would normally be denaturing conditions, and remained electrochemi- cally active for several months. 12 Penicillin acylase (PA) has been adsorbed on to MCM-41 and also by cross linking to silylated MCM-41 using glutaraldeyde as the cross linking agent. The activity of the adsorbed PA was more than five times that of the cross linked enzyme. 25 We have recently shown, by generating adsorption isotherms for cyt c on to a range of MPS, that adsorption is dependent on the silicate pore size and that the peroxidative activity of the adsorbed protein is higher than that of the aqueous protein. 13 Takahashi et al., investigated the immobilization of horserad- ish peroxidase (HRP) and subtilisin on to FSM-16 (folded sheet mesoporous material), MCM-41 (both synthesized using cationic * To whom correspondence should be addressed. Fax: +353-61-202568. Tel: +353-61-202629. E-mail: edmond.magner@ul.ie. 7340 J. Phys. Chem. B 2002, 106, 7340-7347 10.1021/jp0139484 CCC: $22.00 © 2002 American Chemical Society Published on Web 06/27/2002