## 3 Results
### In Situ Dynamic Light Scattering
#### 3.2.3 A Typical Sample: Apomyoglobin  
Apomyoglobin in the molten globule state was investigated by SANS and in situ DLS. Because the protein is not in its native-alike state and the sample concentration is significantly higher than in the living organism, this system is prone to aggregation. Even slight changes in temperature and pH of the environment can lead to sudden aggregation. To ensure that the sample does not change during the SANS experiment, in situ DLS was employed. Every 5 th minute during the hours-long SANS experiment, a DLS measurement was performed.  
The first and the last measurement are shown in Figure 10 and one can see that the sample did not change. The first-order correlation function was evaluated using the CONTIN algorithm [39] and the particle-size distribution according to the scattering intensity can be seen in Figure 11. This confirms that the molten globule apomyoglobin solution did not form more aggregates during the SANS experiment and the amount of aggregates present already at the start of the SANS experiment could be determined. Beyond the R\({}_{\mathrm{H}}\)\(\sim\)2 nm monomers, larger structures of R\({}_{\mathrm{H}}\) -100 nm are present in the solution. Signals of particles of R\({}_{\mathrm{H}}\) > 10 \(\upmu\)m which are attributed to noise can also be identified. Nevertheless, based on the fact that the scattering intensity is strongly dependent on the particle's volume, the largest number of particles in the solution is represented by monomers and the second largest by the R\({}_{\mathrm{H}}\) \(\sim\)100 nm aggregates.  
Figure 11: **Left**: The derived size distribution obtained from the measurements presented in Figure 12. **Right**: close-up of the smaller particles in the solution; besides the monomers with R\({}_{\mathrm{H}}\) \(\sim\)2 nm, larger structures of R\({}_{\mathrm{H}}\) \(\sim\)110 nm are present in the solution. The black line corresponds to the distributions obtained from the first DLS measurement, whereas the blue line indicates the results of the last measurement.  
Figure 10: The electrical field first-order correlation functions of the first and the last DLS measurements performed on the apomyoglobin sample during the neutron beam time. Although larger structures were permanently present in the sample, the two curves nearly coincide. This means that the aggregation state of the sample did not change during the long integration time needed for the SANS measurement. The red lines mark the fit to the data using the CONTIN algorithm.  
Figure 12 shows the corresponding SANS curve to the in situ DLS data discussed above. The scattering profile of the monomeric protein apomyoglobin is represented by the blue line and was determined based on the polymer with excluded volume model [40] and previous knowledge about the protein: excluded volume parameter \(\
u=0.46\) and \(\mathrm{R_{g}}=2.5\) nm [41].  
The green line shows the scattering contribution from the aggregates with \(\mathrm{R_{H}}=150\) nm (which can be roughly approximated to \(\mathrm{R_{g}}=116\) nm, according to the ratio \(\mathrm{R_{g}}/\mathrm{R_{H}}=(3/5)^{0.5}\)[42]). Since for such large morphologies the Guinier regime occurs at lower Q values than the Q-range of the current SANS investigation, this contribution mostly resembles the power law regime of the Beaucage model with the Porod exponent \(p=5/3\)[35]. The red line is obtained by adding the blue and green lines, with an appropriate power law scaling factor at low Q, to describe the data well. Using this additional information from the in situ DLS measurement, one can now interpret the SANS curve better.