Extent Of Cross-link Formation Research Articles

Discovery of a Photoinduced Histidine-Histidine Cross-Link in an IgG4 Antibody

A novel histidine-histidine (His-His) photooxidative cross-link has been identified in an IgG4 antibody. It was formed between the side chain of a histidine residue of the antibody and histidine from the formulation buffer. The structure of the cross-link was elucidated using high performance liquid chromatography (HPLC) hyphenated to tandem mass spectrometry (MS/MS) with higher energy collisional dissociation (HCD). The cross-link was found in multiple conformations, as the location of the oxygen varied. Furthermore, the extent of cross-link formation was shown to correlate with the amount of light the antibody was exposed to as well as the solvent accessibility of each modification site.

Cross-Linking Behavior and Foaming Properties of Bovine α-Lactalbumin after Glycation with Various Saccharides

α-Lactalbumin was glycated via the Maillard reaction in the dry state using various mono- and oligosaccharides. The reaction resulted not only in coupling of the saccharides to α-lactalbumin but also in cross-linked proteins. The glycation rate and the extent of cross-link formation were highly dependent on the saccharide used. Glycation by arabinose and xylose led to a very fast protein cross-link formation, whereas glucose showed a relatively low protein cross-linking ability. The stability of foams, created using the various glycated protein samples, depended on the type of saccharide used, the extent of glycation, and possibly the amount of cross-linked protein. Compared to nonmodified α-lactalbumin, glycation with rhamnose and fucose improved foam stability, whereas application of glucose, galacturonic acid, and their oligosaccharides did not exert a clear effect. Mass spectrometric analysis revealed that dehydration of the Amadori products is an indicator of the formation of protein cross-links.

The excitation of 8-methoxypsoralen with visible light: reversed phase HPLC quantitation of monoadducts and cross-links.

The formation of 8-methoxypsoralen-DNA monoadducts and cross-links is presumed to be responsible for the efficacy of photochemotherapies that employ 8-methoxypsoralen activated with long-wavelength ultraviolet radiation (UVA, 320-400 nm). In this report it is shown that 8-methoxypsoralen can also be activated with visible light (419 nm). Bovine aorta smooth muscle cells were treated with 8-methoxypsoralen (1,000 ng/mL) and 419 nm light (up to 12 J/cm2). Cellular DNA was isolated, hydrolyzed using nucleolytic enzymes and then analyzed by reversed-phase high-performance liquid chromatography. The primary effect of using visible light instead of long-wavelength ultraviolet radiation is a more than 10-fold reduction in the extent of cross-link formation. Because the extent of monoadduct and cross-link formation has not been routinely measured in experiments in which cellular assays have been performed, it is difficult to correlate cell response to the presence of a particular type of 8-methoxypsoralen photoadduct (monoadduct or cross-link). Thus, the use of visible light allows the study of cells containing nearly 100% monoadducts. In addition, the reduction in cross-link formation when visible light is used to activate the compound may also reduce the mutagenicity of 8-methoxypsoralen and hence enhance its therapeutic efficacy.