Book title: Molecules: Nucleation, Aggregation And Crystallization
Editors: Sedzik J, Riccio P
Publisher: World Scientific
Abstract: Using a unique molecular-imprinting method we show in this article that human growth hormone, ribonuclease and myoglobin from horse, lysozyme, hemoglobin and albumin can be adsorbed selectively, indicating that the method may be universal or at least applicable to a great number of proteins. A gel with specific adsorption of three model proteins was synthesized in order to demonstrate that the beds can be employed to remove (traces of) several proteins contaminating a sample ("negative purification"). The degree of selective recognition is high, judging from the fact that myoglobin from horse, but not that from whale, was adsorbed onto a column designed to bind specifically the former protein. This selectivity is noteworthy since these two proteins have similar amino acid sequences and 3-D structures. The method for the synthesis of the specific gels involves polymerization of appropriate monomers (for instance, acrylamide and its derivatives) in the presence of the protein to be adsorbed specifically, granulation of the gel formed, packing a column with the gel particles, washing the column to remove the protein, and finally application of the sample for selective adsorption of the protein present during the polymerization of the monomers. The approach resembles that used for entrapment (immobilization) of proteins for affinity chromatography and somewhat like that for molecular imprinting of small molecules, with the distinct difference that the monomer composition is quite different and thereby the binding mechanism. This mechanism is discussed, for instance, in terms of (i) a new classification system for chromatographic beds based on the number of bonds between the solute and the matrix and the strength of each bond, and (ii) "non-specific bonds" (these bonds are often harmful in conventional chromatography, but we have used them to our advantage). In this classification system, the selective recognition is characterized by a large number of weak bonds. Therefore, so-called functional monomers are not used for the preparation of the gels because they are often charged and, accordingly, give rise to strong electrostatic interactions, i.e. the beds behave to some extent as ion exchangers or matrices for hydrophobic interaction chromatography. In most experiments we have used a polyacrylamide gel with large pores to facilitate diffusion of proteins into and out of the gel granules. When used in chromatography, these soft gels (which can be used repeatedly) allow only rather low flow rates. This problem can be overcome by a new approach to preparing the granules. Potential applications of the selective beds are discussed, as well as future improvements. These beds can be synthesized for selective adsorption also of bio-particles, for instance viruses and bacteria, and in the form of monoliths (continuous beds).
Template and target information: protein, human growth hormone, ribonuclease, myoglobin from horse, lysozyme, hemoglobin, albumin
Author keywords: affinity chromatography, Artificial gel antibodies, entrapment, molecular imprinting, recognition of proteins, selectivity