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.

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Stabilization of proteolytic enzymes, especially by immobilization, is of considerable interest because of their potential applications in medicine and the chemical and pharmaceutical industries. We report here a detailed comparison of two procedures for trypsin immobilization using the same homobifunctional agent, glutaraldehyde, for the purpose of peptide mapping. These methods include covalent coupling either to controlled pore glass (solid support) or via a cross-linking reaction (without any solid support). The immobilized trypsin preparations were characterized by the determination of immobilization efficiency, which ranged from 68 to > 95%, and measurement of apparent kinetic parameters toward a synthetic peptide-like substrate. Batch digestions of whole denaturated human normal adult hemoglobin (HbA) were performed to obtain peptide maps by capillary zone electrophoresis (CZE). Migration time reproducibility of the CZE maps was excellent, with a mean relative standard deviation of 1.5%. Moreover, the two immobilized enzyme preparations showed excellent reproducibility for repeated digestions. Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry was also used for peptide mass mapping of denaturated HbA digested using the two immobilized trypsin preparations. Even though the two immobilized trypsin preparations do not behave identically, similar sequence coverages of 57% and 61% (for the two HbA chains merged) were achieved for the support-based and cross-linked trypsin preparations, respectively.

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The objectives of this study were to determine the prevalence of chronic respiratory symptoms and asthma in 8‐ to 9‐year‐old children in Paris, and to analyze their medical management. This cross‐sectional study was carried out in 1994 on a randomized sample of 3,756 pupils attending Paris public primary schools. The response rate by parents to an initial standardized self‐administered questionnaire was 94.8%. This questionnaire identified 601 children (17%) as having recurrent respiratory symptoms. Of these children, 555 (92.3%) agreed to participate in a follow‐up survey that evaluated their medical management; these children were examined by 73 school doctors of the city of Paris. Prevalence of parent‐reported doctor‐diagnosed asthma was 6.1%. In addition to these 211 children with asthma, 344 other children had recurrent respiratory symptoms: 120 children were “wheezers,” and the remaining 224 children were “coughers.” Among “chesty” pupils not identified as asthmatics, physical education teachers reported exercise‐induced cough or respiratory discomfort in 13.7%, and nearly 14% had a peak expiratory flow 20% lower than the predicted values for age and height. In children identified as asthmatic, 25.3% were not under medical supervision, 55.5% had never performed lung function tests, 63.7% did not receive any prophylactic treatment, and 59.7% were receiving no treatment. Bronchodilator prophylactic medication before exercise was used by only 7% of asthmatics, although physical training teachers noted chest discomfort on exercise in 30.4% of these pupils. These results confirm that children with asthma and participating in this study were less than optimally investigated, were underdiagnosed and undertreated, and their medical management was not optimal. In addition to its epidemiologic value, the study has helped Paris school doctors to advise parents to refer their children to their general practitioner when asthma was suspected or undertreated. Pediatr Pulmonol. 1998; 26:106–112. © 1998 Wiley‐Liss, Inc.