Glutaraldehyde possesses unique characteristics that render it one of the most effective protein crosslinking reagents. It can be present in at least 13 different forms depending on solution conditions such as pH, concentration, temperature, etc. Substantial literature is found concerning the use of glutaraldehyde for protein immobilization, yet there is no agreement about the main reactive species that participates in the crosslinking process because monomeric and polymeric forms are in equilibrium. Glutaraldehyde may react with proteins by several means such as aldol condensation or Michael-type addition, and we show here 8 different reactions for various aqueous forms of this reagent. As a result of these discrepancies and the unique characteristics of each enzyme, crosslinking procedures using glutaraldehyde are largely developed through empirical observation. The choice of the enzyme-glutaraldehyde ratio, as well as their final concentration, is critical because insolubilization of the enzyme must result in minimal distortion of its structure in order to retain catalytic activity. The purpose of this paper is to give an overview of glutaraldehyde as a crosslinking reagent by describing its structure and chemical properties in aqueous solution in an attempt to explain its high reactivity toward proteins, particularly as applied to the production of insoluble enzymes.

The characterization of protein glycosylation can be a complex and time-consuming procedure, especially for prokaryote O-linked glycoproteins, which often comprise unusual oligosaccharide structures with no known glycosylation motif. In this report, we describe a "top-down" approach that provides information on the extent of glycosylation, the molecular masses, and the structure of oligosaccharide residues on bacterial flagella, important structural proteins involved in the motility of pathogenic bacteria. Flagella from four bacterial pathogens, namely, Campylobacter jejuni, Helicobacter pylori, Aeromonas caviae, and Listeria monocytogenes, were analyzed by this top-down mass spectrometry approach. The approach needs minimal sample preparation and can be performed within a few minutes compared to the tedious and often time-consuming "bottom-up" approach involving proteolytic digestion and LC-MS-MS analyses of the suspected glycopeptides. Multiply protonated protein precursor ions subjected to low-energy collisional activation in a quadrupole time-of-flight instrument showed extensive and specific gas-phase deglycosylation resulting in the formation of abundant oxonium ions with very few fragment ions from peptidic bond cleavages. Structural information on individual carbohydrate residues is obtained using a second-generation product ion scan of oxonium ions formed by collisional activation of the intact protein ions in the source region. The four bacterial flagella examined differed not only by the extent of glycosylation but also by the nature of carbohydrate substituents. For example, the flagellin from the Gram-positive bacterium, L. monocytogenes showed O-linked GlcNAc residues at up to 6 sites/protein monomer. In contrast, the three Gram-negative bacterial pathogens C. jejuni, H. pylori and A. caviae displayed up to 19 Ser/Thr O-linked sites modified with residues structurally related to N-acetylpseudaminic acid (Pse5Ac7Ac) and in the case of Campylobacter include a novel N-acetylglutamine substituent on Pse5Am7Ac.

Twenty phenylthlohydantoln-amlno acids (PTH-AA) are separated by micellar capillary electrophoresis In 13.5 min. A low-energy krypton fluoride laser Is used In a thermooptical absorbance detector to produce typical detection limits (3