Native-like structure of proteins at a planar poly(acrylic acid) brush.
Applying ATR-FTIR (attenuated total reflection Fourier transform infrared) and TIRF (total internal reflection fluorescence) spectroscopy, we have studied the secondary structure and aggregation properties of different proteins which are adsorbed at a poly(acrylic acid) (PAA) brush that covers a macroscopically large, planar surface. The PAA brush has been prepared on the surface of an ATR silicon crystal or a quartz plate. The preparation includes the deposition of a thin poly(styrene) film by spin-coating and the transfer of the diblock copolymer poly(styrene)-poly(acrylic acid) onto the hydrophobic film using the Langmuir-Schafer technique. It has been found that the proteins hen egg white lysozyme, bovine serum albumin, bovine alpha-lactalbumin, and bovine insulin adsorb spontaneously at a PAA brush at neutral pD values, albeit to different degrees. The secondary structure of the proteins was estimated from a decomposition of the amide I'-band in the observed ATR-FTIR spectra. Generally, the fractions of secondary structure elements recovered in this way were almost identical to those found when the proteins are native in solution. In addition, the tendency of insulin to form amyloid fibrils has also been tested when the protein is adsorbed at a planar PAA brush. Insulin is known to form amyloid fibrils in solution at low pH values and elevated temperatures. The experiments performed in this study suggest that a PAA brush does not promote fibril formation of insulin. Rather, insulin that is adsorbed at a PAA brush seems to be excluded from fibril formation pathways even at pD = 2 and 60 degrees C, where fibril formation of insulin is triggered in solution. Overall, the results of this study demonstrate that a planar PAA brush may serve as a mild environment for immobilized proteins.
Reichhart
Christian
C
Technische Universitat Dortmund, Fakultat Chemie, D-44221 Dortmund, Germany.
Czeslik
Claus
C
D000180:Acrylic Resins / Q000737:chemistry*; D000327:Adsorption; D000818:Animals; D002417:Cattle; D002645:Chickens; D007328:Insulin / Q000737:chemistry*; D007768:Lactalbumin / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry*; D011137:Polystyrenes / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D013499:Surface Properties; D013997:Time Factors
D000180:Acrylic Resins; D007328:Insulin; D011137:Polystyrenes; D012710:Serum Albumin, Bovine; C006903:carbopol 940; D007768:Lactalbumin; C421864:hen egg lysozyme; D009113:Muramidase