Protein Mass Determination using Small Angle X-Ray Scattering

The molecular weight of Glucose Isomerase was determined with the SAXSpace small-angle X-ray scattering system. The results are in good agreement with theoretically calculated values from X-ray crystallography data.

Protein mass determination by SAXS

Small-angle X-ray scattering is a non-destructive and quick method to study the structure of proteins in solution, i.e. in their native state. The mass of protein particles in solution is an important parameter giving quickly information about the aggregation state of the sample. It therefore can be used as a quality control parameter as it readily identifies undesired denaturation and aggregation of the proteins present in the sample.

Moreover, SAXS is able to identify the degree of oligomerization of proteins in its native environment. Even if the high resolution structure is known from protein crystallography it is not always clear, if it exists as a monomer or an oligomer in its native state.

The determination of the scattered intensity from the SAXS experiment combined with density measurements gives access to the mass and therefore the oligomeric state of the protein particles in solution.

Experimental and Results

Glucose Isomerase from Streptomyces rubiniginosus (1% w/w) was dissolved in 0.1 M Tris buffer at pH 8.0 and was measured with the SAXSpace system in line collimation for 10 min.

The background-subtracted data was brought to an absolute scale using water as a secondary standard. In order to determine the mass of the scattering particles the forward scattering intensity Iabs(0), determined by a Guinier extrapolation, is needed. Furthermore the partial specific volume of the protein v and the electron density difference between protein and buffer Δρ have to be known. They were determined from the protein sequence, the chemical composition of the solvent and the densities of sample d and solvent  d2 measured by a DMA 5000 density meter from Anton Paar.

The mass determined by SAXS and the theoretical value of a tetramer calculated from the sequence available from the Protein Data Bank agree well. This proofs that the protein is present in solution as a tetramer. 

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