Glutaraldehyde has been used for several decades as an effective crosslinking agent for many applications including sample fixation for microscopy, enzyme and cell immobilization, and stabilization of protein crystals. Despite of its common use as a crosslinking agent, the mechanism and chemistry involved in glutaraldehyde crosslinking reaction is not yet fully understood. Here we describe feasibility study and results obtained from a new approach to investigate the process of protein crystals stabilization by glutaraldehyde crosslinking. It involves exposure of a model protein crystal (Lysozyme) to glutaraldehyde in alkaline or acidic pH for different incubation periods and reaction arrest by medium exchange with crystallization medium to remove unbound glutaraldehyde. The crystals were subsequently incubated in diluted buffer affecting dissolution of un-crosslinked crystals. Samples from the resulting solution were subjected to protein composition analysis by gel electrophoresis and mass spectroscopy while crosslinked, dissolution resistant crystals were subjected to high resolution X-ray structural analysis. Data from gel electrophoresis indicated that the crosslinking process starts at specific preferable crosslinking site by lysozyme dimer formation, for both acidic and alkaline pH values. These dimer formations were followed by trimer and tetramer formations leading eventually to dissolution resistant crystals. The crosslinking initiation site and the end products obtained from glutaraldehyde crosslinking in both pH ranges resulted from reactions between lysine residues of neighboring protein molecules and the polymeric form of glutaraldehyde. Reaction rate was much faster at alkaline pH. Different reaction end products, indicating different reaction mechanisms, were identified for crosslinking taking place under alkaline or acidic conditions.

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Protein crystals, routinely prepared for the elucidation of protein 3D structures by X-ray crystallography, present an ordered and highly accurate 3D array of protein molecules. Inherent to the 3D arrangement of the protein molecules in the crystal is a complementary 3D array of voids made of interconnected cavities and exhibiting highly ordered porosity. The permeability of the porosity of chemically crosslinked enzyme protein crystals to low molecular weight solutes, was used for enzyme mediated organic synthesis and size exclusion chromatography. This permeability might be extended to explore new potential applications for protein crystals, for example, their use as bio-templates for the fabrication of novel, nano-structured composite materials. The quality of composites obtained from "filling" of the ordered voids in protein crystals and their potential applications will be strongly dependent upon an accurate preservation of the order in the original protein crystal 3D array during the "filling" process. Here we propose and demonstrate the feasibility of monitoring the changes in 3D order of the protein array by a step-by-step molecular level monitoring of a model system for hydrogel bio-templating by glutaraldehyde crosslinked lysozyme crystals. This monitoring is based on step-by-step comparative analysis of data obtained from (i) X-ray crystallography: resolution, unit cell dimensions and B-factor values and (ii) fluorescence decay kinetics of ultra-fast laser activated dye, impregnated within these crystals. Our results demonstrated feasibility of the proposed monitoring approach and confirmed that the stabilized protein crystal template retained its 3D structure throughout the process.

Directed deposition of silver on the surface of single, soluble enzyme molecules was made possible by controlled conjugation of new silver-reducing moieties to the surface of the enzyme molecule and their reaction with silver ions. Molecular enzyme-silver hybrids carrying low or high crystalline silver deposited on their surface were obtained, retaining their solubility and enzymatic activity. The feasibility of "wiring" of the active site of glucose oxidase-silver hybrid thus obtained to platinum electrode was readily demonstrated enabling glucose determination in the absence of oxygen.Electroless deposition of metals has many applications in fabrication on the basis of micro-and nanotechnologies, for example, interconnects and packaging affected by Cu, Co, Ni, and Ag high-quality ultrathin films, deposited on solid substrates. 1 In recent years, several attempts were made to adopt electroless deposition technologies practiced in the microelectronics industry for the fabrication of new hybrids made of biological templates, for example, DNA and protein arrays for the synthesis of interconnects. Within this context, metallization by electroless deposition of protein-made natural arrays was recently successfully demonstrated for the fabrication of nanowires from microtubules, 2-6 viral envelopes, 7-9 amyloid fibers, 10 and actin. 11 These metallizations were directed to the substrate's surface by the adsorption of palladium or platinum ions followed by their chemical reduction 2-5,7-9 or by surface labeling with colloidal gold particles. 10,11 Enlargement of the nucleation sites thus obtained into continuously deposited metallic films was carried out by immersion in plating solution containing the metal ions of choice such as Ag +1 or Ni +2 and reducing agents such as NaBH 4 or dimethylaminoborane. This nucleation/growth mechanism led to the formation of relatively thick metal deposits, for example, 10-35 nm. 4 Here, we propose and demonstrate feasibility of a novel mechanism for directing silver metallization to the surface of single, soluble enzyme molecule, enabling the construction of a very thin metallic film on its surface, with retention of its biological activity and solubility. Such soluble hybrids of active enzymes are expected to provide novel molecular tools for applications requiring "wiring" of nanosized sensors to electrodes and composite biochips. 12 The new method is based on coupling of a reducing agent to the surface of soluble enzyme molecules, followed by removal of nonbound reducer. Addition of ion metals will lead to the formation of nucleation sites accompanied by local growth leading to the formation of metallic "patches". The density and continuity of these patches may be controlled by the density of the reducing moieties, displayed on the surface of the enzyme as monolayered or multilayered arrays. This process should result in the formation of metallic coating stretching out close enough to the catalytic site of the enzyme without blocking it to substrate access.The enzyme selected for f...