Reversible denaturation of Brazil nut 2S albumin (Ber e1) and implication of structural destabilization on digestion by pepsin.
The high resistance of Brazil nut 2S albumin, previously identified as an allergen, against proteolysis by pepsin was examined in this work. Although the denaturation temperature of this protein exceeds the 110 degrees C at neutral pH, at low pH a fully reversible thermal denaturation was observed at approximately 82 degrees C. The poor digestibility of the protein by pepsin illustrates the tight globular packing. Chemical processing (i.e., subsequent reduction and alkylation of the protein) was used to destabilize the globular fold. Far-UV circular dichroism and infrared spectroscopy showed that the reduced and alkylated form had lost its beta-structures, whereas the alpha-helix content was conserved. The free energy of stabilization of the globular fold of the processed protein as assessed by a guanidine titration study was only 30-40% of that of the native form. Size exclusion chromatography indicated that the heavy chain lost its globular character once separated from the native 2S albumin. The consequences of these changes in structural stability for degradation by pepsin were analyzed using gel electrophoresis and mass spectrometry. Whereas native 2S albumin was digested slowly in 1 h, the reduced and alkylated protein was digested completely within 30 s. These results are discussed in view of the potential allergenicity of Brazil nut 2S albumin.
Koppelman
Stef J
SJ
TNO Nutrition and Food Research, Zeist, The Netherlands. koppelman@voeding.tno.nl
Nieuwenhuizen
Willem F
WF
Gaspari
Marco
M
Knippels
Leon M J
LM
Penninks
André H
AH
Knol
Edward F
EF
Hefle
Susan L
SL
de Jongh
Harmen H J
HH
D055516:2S Albumins, Plant; D000418:Albumins / Q000737:chemistry* / Q000378:metabolism*; D052179:Antigens, Plant; D031383:Bertholletia / Q000737:chemistry*; D010434:Pepsin A / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism*
D055516:2S Albumins, Plant; D000418:Albumins; D052179:Antigens, Plant; D010940:Plant Proteins; D011498:Protein Precursors; C103364:2S albumin, brazil nut; D010434:Pepsin A