4.1 Proteins and polymers under MAS
4.1.2 Experiments with proteins
In the previous chapter it was discussed that not only the molecular weight, but also the shape of the molecules is important when performing SEC studies. This issue is of great importance when studying proteins. Proteins are formed by sequences of monomers called amino acids that undergo condensation reactions releasing water and forming a peptide bond. To be able to complete their biological functions this sequences fold into different spatial conformations driven by different intermolecular forces such as hydrogen bonds or van der Waals forces, the final spatial conformations vary from tubular proteins to globular ones [151]. Therefore, it is possible that a protein with a lower molecular weight could diffuse slower because of a larger spatial conformation.
In order to appreciate this effect the diffusion coefficients of a group of different proteins were recorded with high concentrations of urea and without urea at a 2 kHz spinning rate. Urea is an organic compound that in concentrations between 8 and 10 M is a powerful protein denaturant as it disrupts the noncovalent interactions in proteins [152]. Therefore, proteins unfold and increase their effective hydrodynamic size reducing their diffusion coefficient. The results are shown in table 4.1
Proteins
Table 4.1: Summary of diffusion coefficients of samples of 2 mM proteins of varying molecular weights in buffer with and without urea 8 M at 2 kHz spinning rate
Looking at the table above, the data is consistent with the statement that urea can disrupt the noncovalent interactions and expand the spatial conformation of the proteins increasing their effective radius and consequently decreasing the diffusion coefficient. In addition, the result are also in agreement with the importance of protein conformation after the folding process that was mentioned before. This conformation can affect to the diffusion as it is shown by one of the largest proteins of the studied set, the albumin from chicken egg, which it is also the fastest one.
Although the proteins used in this experiments have similar weights to the polymers used in previous studies [75] the diffusion coefficient that they show were larger than the polymer ones which were diffusing between 3.5 and 4.5 × 10-10 m2 s-1. This is in agreement with the fact that polymers show larger sizes than proteins due to the protein folding, as the results became more similar when the urea was used. However, the concentration used for the proteins was much lower than the used for the polymers which could contribute to a slower diffusion coefficient for the polymers.
Nonetheless, the result were not exactly in agreement with the range of velocities expected because the proteins diffused 10x faster than the polymers in static conditions. Therefore, as it was mentioned before, the high spinning rate is increasing the diffusion coefficient probably due to an increase of the kinetic energy. However, for the proteins studied, the diffusing behaviour of the proteins followed the expected pattern. Therefore, they seem as good candidates to carry on with the diffusion studies under MAS conditions.
Finally, in order to compare the proteins results with the polymer results of similar molecular weights, the chosen proteins to continue the studies were the proteins shown in table 4.2 because they have similar weight to the polymers that were used in our previous studies [75].
Protein Molecular weight (Da)
Bovine Serum Albumin (BSA) 66500
α-Chymotrypsinogen A 25600
β-Lactoglobulin 18400
Bovine α-Lactalbumin 14178
Table 4.2: Summary of proteins used for HR-MAS NMR chromatography
The diffusion coefficients of the proteins were recorded under 2 kHz MAS conditions from samples of 2mM concentration in buffer with and without Sephadex G50. In order to fill the whole rotor with stationary phase the samples were prepared as described in chapter 2. The protein diffusion results are shown in figure 4.5.
Figure 4.5: Diffusion of proteins in buffer at 2 kHz spin rate with and without Sephadex G50
Without the presence of stationary phase, the results shown by the proteins followed the expected pattern, which is, the larger the protein the slower the diffusion coefficient is. The only not expected results is the high speed of the diffusion coefficient compared to the tube results.
When the stationary phase was added, the diffusion coefficient of the smaller molecules was reduced more than the larger ones. This observation is in agreement with size exclusion principles, as the smaller the molecule is the one that can explore more the pores of the stationary phase allowing a greater interaction between the molecule and the stationary phase.
It could also be observed that all the proteins presented a very similar diffusion coefficient in presence of the stationary phase. Although this result could be possible it was unexpected that even the molecules that would not fit in the pores and therefore their diffusion coefficient should barely vary, presented and large variation in the diffusion coefficient (BSA varied from 5.5 to 2.1 × 10-10 m2 s-1 when the pore size max is 30 kDa for globular proteins and this protein is 66.5 kDa see tables 2.1 and 4.2). These two facts together suggested that what the molecules were experiencing was not a size exclusion effect but hindered diffusion due to the large centrifugal force generated in the solution that prevented the molecules from diffusing freely may be due to interferences between them and also with the stationary phase. Therefore, the possible explanation is the fact that such a high spin rate could cause sedimentation effects when the stationary phase was present in the sample, which prevented the molecules of diffusing normally in the solution. However, further studies must be done to support this idea such as putting the sample under high centrifugal forces and see if there is a gradient of concentrations generated.