Enzymatic cross-linking of beta-lactoglobulin: conformational properties using FTIR spectroscopy.
In this study, we use FTIR spectroscopy to probe the conformational changes of beta-lactoglobulin (beta-LG)-the main constituent of whey proteins-as subjected to enzymatic cross-linking by transglutaminase. We investigate both the amide I region (1600-1700 cm(-1)) and the C-H stretching region (2800-3100 cm(-1)). In the amide I region, spectra of denatured conformations of beta-LG, known to be necessary for cross-linking, differ according to the denaturation procedure, i.e., chemical or thermal treatment. Denaturation by chemical denaturants, dithiothreitol (DTT) or beta-mercaptoethanol, show no effect on the alpha-helix, while shifting the monomer dimer equilibrium toward higher monomer concentration. On the other hand, denaturing by thermal treatment dissociates the beta-sheets in the native structure, leading to new intermolecular beta-sheets being formed. Preheated then enzyme cross-linked beta-LG molecules show very similar spectra in the amide I region to the molecules with no cross-linking, indicating minimal effects of the cross-links on the carbonyl stretching mode. However, chemically denatured (using beta-mercaptoethanol) then enzyme cross-linked beta-LG molecules show noticeable diminution in the alpha-helix band and formation of strong hydrogen-bonded intermolecular beta-sheets. In the C-H stretching region, preheated then enzyme cross-linked beta-LG molecules exhibit a different degree of exposure of aliphatic amino acids due to the enzyme action. The same behavior is observed for DTT-treated then enzyme cross-linked beta-LG molecules. Generally, the changes in the C-H stretching region clearly indicate that hydrophobic interactions are altered upon enzymatic cross-linking.
Eissa
Ahmed S
AS
Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695-7905, USA.
Puhl
Christa
C
Kadla
John F
JF
Khan
Saad A
SA
D000577:Amides / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry*; D007782:Lactoglobulins / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012680:Sensitivity and Specificity; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D011503:Transglutaminases / Q000737:chemistry*
D000577:Amides; D003432:Cross-Linking Reagents; D007782:Lactoglobulins; D011503:Transglutaminases