Compuestos sintagmáticos

In the atomization process in CO2 spray drying [23], the CO2

comes in contact with water at the surface of droplets (CO2/water

interface). As supercritical CO2 is non-polar, the protein in the droplets

will be exposed to the hydrophobic CO2 at the droplet interface. Under

these conditions, the protein may reorient, to expose the hydrophobic heme pocket of myoglobin towards to this surface, leading to protein unfolding at the droplet interface. In additional to this surface effect, the drying of the droplets occurs by the mass transfer of water into the CO2

phase as well as the diffusion of CO2 into the droplets. It is the latter that

has been shown to cause acidification of the protein formulation, which also led to protein denaturation as shown by changes in the UV-Vis and CD spectra [23]. However, given that the CO2 spray drying process takes

place in a sealed high pressure vessel, it is not possible to determine whether the CO2/water interface or the acidification is the predominant

factor influencing the resultant protein destabilization.

It was anticipated that the gas bubbling study with CO2 and N2

would help to differentiate the influence of these two factors on the destabilization and aggregation of myoglobin. Although this method uses the myoglobin solution as the bulk phase (in contrast to the supercritical CO2 being the bulk phase in the case of the spray drying

process), the bubbles will still create a gas/water interface that may be representative of the behaviour observed during spraying. Moreover,

the bubbling method has previously been shown to enhance the protein aggregation rate 40 times compared to stirring, due to rapid regeneration and renewal of interfaces [3]. Furthermore, bubbling with CO2 was expected to result in the acidification of myoglobin solutions in

a similar manner to what was observed during the high pressure spray drying process, as CO2 reacts with the water to form carbonic acid. A

similar pH shift has previously been observed for a milk solution (120 g in 1000 g water), where the pH was also found to decrease from 6.8 to about 5 when exposed to high pressure CO2 under static conditions at

20-40 bar [29]. In contrast, the solutions exposed to N2 bubbling were not

expected to show any pH shift, as N2 does not form an acid in water.

As anticipated, bubbling with N2 resulted in no change in the pH,

while bubbling with CO2 showed a shift in pH to values lower than 5.0 for

all the myoglobin solutions, except those with the starting pH of 4.0. However, only the myoglobin solution prepared at pH 4.5 showed a change in the α-helix and heme binding of myoglobin (as observed by UV/Vis and CD at the Soret band), which was due to the shift in pH to 4.3. These results suggest that the heme experiences partial dislocation or dissociation within the myoglobin upon reducing the pH [30-32]. For this same solution, however, no change in the fluorescence signal was observed after bubbling, suggesting that the heme remains within the heme pocket. Based on these observations, it would seem that myoglobin is in an acid-denatured state under low pH conditions, and that the structure resembles a molten globule [33-35]. Additionally, myoglobin may present as an intermediate form between the native heme-bound and heme-dissociated forms at pH below 4.5 [33, 36].

In contrast, the fluorescence data for the myoglobin solution with a starting pH of 4.0 indicates that there is a clear change in the heme binding (Fig. 5). In general, an increase in the fluorescence signal indicates that the myoglobin structure is less quenched by heme [33, 37- 39]. This is also supported by the A409/A280 ratio, which decreases when

the heme binding to the proximal histidine at the heme pocket is changed [40, 41]. For the completely heme-free form of myoglobin (apomyoglobin) extracted by acid-methylethylketone [42], the ratio of fluorescence intensity to protein absorption (I/A280) is higher than that of

myoglobin [23]. It is often assumed that any increase in the fluorescence signal corresponds to a loss of heme. However, it has also been shown that the intermediate form of myoglobin, which allows solvent molecules (typically water) to access the tryrosyl residues of tryptophan, can also result in an increase in the fluorescence signal [33]. Moreover, Stiebler et al. [43] reported that about 0.5 nmol/ml of heme was soluble in acidified aqueous solution (0.5 M sodium acetate buffer pH 4.8), which would correspond to about 0.0005% of the total heme in the myoglobin

solutions before CO2 bubbling. Taking all this into consideration, it

appears that destabilization of the heme binding of myoglobin that occurs at pH 4 is most likely the result of the presence of the intermediate form of myoglobin. Moreover, it is expected that this acid denaturation will be reversible, as no heme will be lost from the myoglobin structure [44].

From this study, it can be concluded that if the pH is lower than 4.5 (either from the starting pH or induced by the formation of carbonic acid), the myoglobin undergoes acid denaturation that results in structural changes. In contrast, the gas/water interface has no influence the myoglobin structure.

4.2 Effect of gas bubbling on the formation of myoglobin aggregates