15B-8 |
Fourier transform infrared spectroscopy (FT-IR) determination of ligand-induced secondary and tertiary structural changes in bovine plasminogen |
K. D. HAYES, B. F. Ozen, and L. J. Mauer. Department of Food Science, Purdue University, 1160 Food Science Building, West Lafayette, IN 47907-1160 Human PG undergoes a large tertiary structural change in the presence of lysine derivatives (e.g. e-amino caproic acid, EACA). This change facilitates human PG activation by human PG activators, resulting in elevated plasmin levels. This structure-function relationship is thought to be conserved in bovine PG. Bovine PG is the precursor of the native milk protease plasmin, and is initially present in much higher concentrations. a-Casein stimulates PG activation, and plasmin activity influences cheese ripening. Lysine binding sites on casein micelles may be critical for regulating PG activation in dairy products. The objectives of this study were to determine secondary and tertiary structures of bovine PG in the presence and absence of EACA. Spectra of EACA+PG, EACA, and PG in water and deuterium were collected by FT-IR with horizontal ZnSe-ATR and a MCTA detector. Second derivative and Fourier-self deconvoluted spectra of [(PG+EACA)-EACA] and [PG- water (or deuterium)] in the amide I region (1700-1600 cm-1) were used to detect changes in secondary structure of PG after EACA addition. Change in PG tertiary structure was determined by comparing ratios of amide II to amide I bond intensities. Comparing second derivative spectra revealed changes in the secondary structure of PG after the addition EACA. Bands in the amide I region shifted, rearranged, or dampened, and a new peak was observed at 1656 cm-1 (corresponding to a-helical structures). Changes were more obvious in water than deuterium. After 0.5 h of deuteration, amide II/amide I intensities were 0.8 and 0.76 for PG+EACA and PG, respectively. These observations show that both secondary and tertiary structural changes were induced in PG by EACA. Data were consistent with human PG studies using neutron scattering (tertiary structure) and circular dichroism (secondary structure) methods. FT-IR was able to monitor both secondary and tertiary structural changes in bovine PG resulting from ligand binding.
Session 15B, Dairy Foods: Chemistry, microbiology and sensory analysis of various cheeses
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