Conformational stability of alpha-lactalbumin missing a peptide bond between Asp66 and Pro67.
The peptide bond between Asp66-Pro67 of alpha-lactalbumin was cleaved with formic acid (cleaved alpha-lactalbumin). Secondary structural changes of the cleaved alpha-lactalbumin, in which the two separated polypeptides were joined by disulfide bridges, were examined in solutions of sodium dodecyl sulfate (SDS), urea, and guanidine hydrochloride. The structural changes of the cleaved alpha-lactalbumin were compared with those of the intact protein. The relative proportions of secondary structures were determined by curve fitting of the circular dichroism. The cleaved alpha-lactalbumin contained 29% alpha-helical structure as against 34% for the intact protein. Some helices of the cleaved alpha-lactalbumin which had been disrupted by the cleavage appeared to be reformed upon the addition of SDS of very low concentration (0.5 mM). In the SDS solution, the helicities of both the intact and cleaved proteins increased, attaining 44% at 4 mM SDS. On the other hand, the helical structures of the cleaved alpha-lactalbumin began to be disrupted at low concentrations of guanidine hydrochloride and urea compared with that of the intact protein. However, no difference was observed in the thermal denaturations of the intact and cleaved proteins, except for the difference in the original helicities. The helicities of both proteins decreased with an increase of temperature up to 65 degrees C and recovered upon cooling.
Hamada
S
S
Department of Applied Chemistry, Okayama University of Science, Japan.
Moriyama
Y
Y
Yamaguchi
K
K
Takeda
K
K
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D002942:Circular Dichroism; D004220:Disulfides; D004355:Drug Stability; D000431:Ethanol; D005561:Formates; D019791:Guanidine; D006146:Guanidines; D007768:Lactalbumin / Q000737:chemistry*; D011392:Proline; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014508:Urea
D004220:Disulfides; D005561:Formates; D006146:Guanidines; C030544:formic acid; D001224:Aspartic Acid; D000431:Ethanol; D014508:Urea; D007768:Lactalbumin; D011392:Proline; D019791:Guanidine