Interfacial Behavior of N-Nitrosodiethylamine/Bovine Serum Albumin Complexes at the Air−Water and the Chloroform−Water Interfaces by Axisymmetric Drop Tensiometry

Abstract

Interfacial properties of N-nitrosodiethylamine/bovine serum albumin (NDA/BSA) complexes were investigated at the air-water interface. The interfacial behavior at the chloroform-water interface of the interaction product of phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), dissolved in the chloroform phase, and NDA/BSA complex, in the aqueous phase, were also analyzed by using a drop tensiometer. The secondary structure changes of BSA with different NDA concentrations were monitored by circular dichroism spectroscopy at different pH and the NDA/BSA interaction was probed by fluorescence spectroscopy. Different NDA/BSA mixtures were prepared from 0, 7.5 x 10(-5), 2.2 x 10(-4), 3.7 x 10(-4), 5 x 10(-4), 1.6 x 10(-3), and 3.1 x 10(-3) M NDA solutions in order to afford 0, 300/1, 900/1, 1 500/1, 2 000/1, 6 000/1, and 12 500/1 NDA/BSA molar ratios, respectively, in the aqueous solutions. Increments of BSA alpha-helix contents were obtained up to the 2 000/1 NDA/BSA molar ratio, but at ratios beyond this value, the alpha-helix content practically disappeared. These BSA structure changes produced an increment of the surface pressure at the air-water interface, as the alpha-helix content increased with the concentration of NDA. On the contrary, when alpha-helix content decreased, the surface pressure also appeared lower than the one obtained with pure BSA solutions. The interaction of DPPC with NDA/BSA molecules at the chloroform-water interface produced also a small, but measurable, pressure increment with the addition of NDA molecules. Dynamic light scattering measurements of the molecular sizes of NDA/BSA complex at pH 4.6, 7.1, and 8.4 indicated that the size of extended BSA molecules at pH 4.6 increased in a greater proportion with the increment in NDA concentration than at the other studied pH values. Diffusion coefficients calculated from dynamic surface tension values, using a short-term solution of the general adsorption model of Ward and Tordai, also showed differences with pH and the NDA concentration. Both, the storage and loss dilatational elastic modulus were obtained at the air-water and at the chloroform-water interfaces. The interaction of NDA/BSA with DPPC at the chloroform-water produced a less rigid monolayer than the one obtained with pure DPPC (1 x 10(-5) M), indicating a significant penetration of NDA/BSA molecules at the interface. At short times and pH 4.6, the values of the storage elastic modulus were larger and more sensible to the NDA addition than the ones at pH 7.1 and 8.4, probably due to a gel-like network formation at the air-water interface.

Interaction of N-nitrosodiethylamine/bovine serum albumin complexes with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine monolayers at the air–water interface

“…As demonstrated in Ref. [30], this NDA/BSA molar ratio shows a higher α-helix proportion of the BSA structure. The smaller area increments at 20 mN/m, compared with the ones at 10 mN/m, are probably caused by the proximity of the BSA maximum surface pressure [35] (around 24 nM/m), which can contribute to enhance the squeezing out of protein or DPPC molecules.…”

“…This behavior could be representative of a relatively larger monolayer penetration by NDA/BSA molecules at higher pressures or it could simply be a consequence of the NDA/BSA complex having a larger size, as was recently demonstrated [30]. Furthermore, it could also be due to significant changes in the secondary structure of BSA, produced by the interaction with NDA molecules [30]. This is, BSA should be looser when penetrating the DPPC monolayer.…”

“…Table 1 shows the peak values of C −1 s at the onset of this new phase for the different NDA/BSA ratios used. We notice that the presence of NDA in the BSA molecules, interacting with DPPC at the airwater interface, produces more rigid films when the NDA/BSA molar ratio is 2000, probably caused by the high BSA α-helix content [30].…”

“…This variation can be interpreted as the penetration of the DPPC monolayer by BSA. Assuming that whole BSA molecules penetrate the DPPC monolayer with an average hydrodynamic radius of 4 nm at the air-water interface [30], we estimate from the above molecular area difference that there is about 1 BSA molecule per 6×10 4 DPPC molecules in the monolayer complex.…”

“…In recent experiments [30], we have investigated the interactions between N -nitrosodiethylamine (NDA) and BSA at the air-water and chloroform-water interfaces using axisymmetric drop tensiometry, fluorescence, and circular dichroism. There, we demonstrated that different NDA concentrations change the BSA secondary structure and consequently the hydrophobic and elastic BSA properties in interfaces.…”