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How to detect Free Chromophore?

Background information: Flow Induced Dispersion Analysis is a capillary-based microfluidic system utilising Taylor Dispersion to determine hydrodynamic radius, Rh. In brief, flow rate in the centre of the capillary is faster than that adjacent to capillary wall. The resulting radial concentration gradients at the front and the tail of the dispersion results in diffusion of indicator molecule, which enables a “first principle” biophysical measurement of size.

All detectible molecules will exhibit similar Taylorgram in FIDA, i.e., most molecules ’Taylorgram will center around 110 seconds in most assays, unless viscosity is not the same as pure water.  If there are multiple detectable species in a solution, their Taylorgram will be superimposed on top of each other’s. One common example which is encountered in chemical labelling of biomolecules.  After labelling, the unreacted free dye isremoved using a desalting column. However, one step desalting columns often leaves some free dye in thesample.

FIDA can be used to measure the percent of free dye present in a sample.  Although all detectable molecules in a sample travel at the same time and their signal superimposed, Fida software can distinguish the two Taylorgrams. The smaller dye molecule has a sharper Taylorgram and its size is approximately 0.6 nm, and thus the software can fit the raw data with two species, keeping one species size at 0.6 nm (Figure 1).  If there are in fact two species present in the sample, the software will find them. Software will also indicate the percent of each species in the sample.If there is only one species present, the software will indicate that by reporting only one size.

Figure 1: A) Single species fit
B) Two species fit: one species, dye, fixed at 0.6 nm

For example, Figure 2 A shows a Protein X sample which has been fitted both to single and two species.  Both fits give similar sizes, however, two species fit detects 1% free dye present.  Figure 2 B shows a mutant of Protein X fitted to both single and double species.  In this case, two species fit detects 10% free dye presence in this sample.  Notice that this sample exhibits sharper peak, relative to the wild type in A, indicating presence of a smaller detectable molecule.  Rh with two species fit is in better agreement with the Rh of the wild type in A, which is more reasonable.
With practice, it is relatively easy to spot presence of free dye, if the percentage of free dye is over five, such as in this example. However, lower percentage of free dye is harder to spot with the naked eye.

Figure 2: A) protein X, fitted to both 1 and 2 species.
Only 1% free dye is detected and both fits give similar sizes
B) A mutant of protein X, where 10% free dye is detected. Two species fit size is in better agreement with wild type A.

The best remedy for a sample with excess free dye is to perform another desalting step or to use a column with dye affinity resin.

Although it is preferable to work with samples that have zero or less than 2 percent free dye, it is possible to work with samples with higher free dye.  In circumstances where further desalting results in an unacceptable protein loss, such samples could still be used by applying two species fit. In these cases, it is recommended to use the Rh values from the two species fit for affinity and conformational change determination.  Up to five percent of free dye is acceptable.