Effects of physicochemical factors on the secondary structure of beta-lactoglobulin.
Fourier transform infrared spectroscopy and differential scanning calorimetry were used as complementary techniques to study changes in the secondary structure of beta-lactoglobulin under various physicochemical conditions. The effects of pH (3-9), NaCl (0-2 M), and lactose, glucose and sucrose (100-500 g/l) in the temperature range 25-100 degrees C on the conformation sensitive amide I band in the i.r. spectrum of beta-lactoglobulin in D2O solution were examined. The 1692 cm-1 band in the amide I band profile had not been definitively assigned in previous studies of the i.r. spectrum of beta-lactoglobulin. The decrease in this band at ambient temperature with time or upon mild heating was attributed to slow H-D exchange, indicating that it was due to a structure buried deep within the protein. The disappearance of the 1692 cm-1 band on heating was accompanied by the appearance of two bands at 1684 and 1629 cm-1, assigned to beta-sheets. The 1692 cm-1 band was therefore attributed to a beta-type structure. beta-Lactoglobulin showed maximum thermal stability at pH 3 and was easily denatured at pH 9. On denaturation, the protein unfolded into more extensive random coil structures at pH 9 than at pH 3. After 10 h at pH 9 (25 degrees C), beta-lactoglobulin was partly denatured. Heating to 60-80 degrees C generally resulted in the loss of secondary structure. At all pH values studied, two new bands at 1618 and 1684 cm-1, characteristic of intermolecular beta-sheet structure and associated with aggregation, were observed after the initial denaturation. Differential scanning calorimetry studies indicated that the thermal stability of beta-lactoglobulin was enhanced in the presence of sugars. The Fourier transform i.r. results obtained provide evidence that sugars promoted the unfolding of beta-lactoglobulin via multiple transition pathways leading to a transition state resisting aggregation.
Boye
J I
JI
Department of Food Science and Agricultural Chemistry, Macdonald Campus of McGill University, Ste-Anne-de-Bellevue, Montreal, Québec, Canada.
Ismail
A A
AA
Alli
I
I
D002152:Calorimetry, Differential Scanning; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003903:Deuterium; D005947:Glucose / Q000494:pharmacology; D006358:Hot Temperature; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry*; D007785:Lactose / Q000494:pharmacology; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012965:Sodium Chloride / Q000494:pharmacology; D017550:Spectroscopy, Fourier Transform Infrared; D013395:Sucrose / Q000494:pharmacology
D007782:Lactoglobulins; D012965:Sodium Chloride; D013395:Sucrose; D006859:Hydrogen; D003903:Deuterium; D005947:Glucose; D007785:Lactose