Mechanism of bovine serum albumin aggregation during ultrafiltration.
Protein fouling is a critical problem for ultrafiltration. In this study, we adopted bovine serum albumin (BSA) as a model protein and polysulfone membrane as a typical ultrafiltration membrane. We then investigated the factors of the protein denaturation and aggregation, such as stirring shear stress and intermolecular exchange of disulfide during ultrafiltration, and discussed the BSA fouling mechanism. Fourier transform-infrared analysis revealed that magnetic stirring did not cause any difference in the secondary structural change of BSA gel-like deposits on the ultrafiltration membrane. BSA aggregates were collected from BSA gel-like deposits on the ultrafiltration membrane by centrifugation. Polyacrylamide gel electrophoresis in SDS analysis of BSA aggregates proved that the major binding of the BSA aggregates involved intermolecular disulfhydryl binding and that capping the free thiol group in BSA molecules with cysteine induced a remarkable decrease in the amount of the BSA aggregates during ultrafiltration. We concluded that one of the main factors in the BSA aggregation during ultrafiltration is the intermolecular exchange of disulfide through cysteinyl residue. We also found that the BSA aggregation caused a decrease in alpha-helix from 66% to 50% and an increase in beta-sheet from 20% to 36%, which was presumably because the cysteine residues associated with the intermolecular disulfide bonds had been located in alpha-helices.
Maruyama
T
T
National Food Research Institute, Kannondai 2-1-2, Tsukuba, Ibaraki, Japan 305-8642. mnaka@nfri.affrc.go.jp
Katoh
S
S
Nakajima
M
M
Nabetani
H
H
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003545:Cysteine / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008623:Mercaptoethanol / Q000494:pharmacology*; D008956:Models, Chemical; D011485:Protein Binding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D012996:Solutions; D017550:Spectroscopy, Fourier Transform Infrared; D014462:Ultrafiltration
D012996:Solutions; D012710:Serum Albumin, Bovine; D008623:Mercaptoethanol; D003545:Cysteine