Even the most primitive cells perform a wide range of extremely complex functions. Despite all that has been learned, scientists are only beginning to understand how they do it.
A major hinderance to this understanding is the fact that cells are complex architectures. A relevant example is the Golgi apparatus, a cellular organelle (specialized cellular subunit) where many proteins are chemically modified after synthesis.
The primary biochemical modification that occurs here is the addition of sugar units to proteins. Affixing sugar units on proteins enables cells to ward off foreign invaders, communicate with other cells, and many other important functions.
Heparin sulfate is one protein that is heavily modified with sugar units in the Golgi apparatus. It is a very important protein that enables the body to regulate blood coagulation, tumors to proliferate, and other medically-relevant life processes.
How the Golgi apparatus synthesizes different chemical variants of heparin sulfate, with different molecular architectures and functions, is not well understood. Robert Linhardt (Rensselaer Polytechnic Institute, New York) and coworkers have worked towards addressing this question.
The microfluidic chip.
The device the scientists utilized to probe the biochemical modification of the heparin sulfate protein is a microfluidic chip. This is a small-scale device that enables one to rapidly perform many different chemical reactions on a small scale.
With an appropriate design, different chemical solutions can be isolated from or mixed with one another. A description of the device design follows.
The device consists of a central channel, with multiple separate reservoirs placed alongside it. Each reservoir is connected to the central channel by a small channel of its own; think of a hallway in a house, with different rooms leading off from the main hallway.
Different solutions can be spotted into the separate reservoirs. The solution droplets remain in their separate reservoirs, without mixing with solutions in other reservoirs, unless a voltage is applied (why a voltage mixes the solutions will be discussed later).
By applying a voltage to an electrode adjacent to one of the solutions in a given reservoir, one droplet can be moved from its reservoir to a mixing area (in this research, at a rate of approximately 3 millimeters per minute). In the mixing area, two droplets that were initally from separate reservoirs can be mixed together.
After mixing (and a biochemical reaction, if the solutions contain enzyme molecules), another voltage can be applied, to separate the mixed droplet into two droplets of equal size. One of the droplets can be taken off of the chip for chemical analysis, and the other can be sent on to participate in further chemical reactions.
Thus, the scientists' device enables one to initiate biochemical reactions on a small scale, and analyze the resulting products of the reactions. A noteworthy advantage is that many reactions can be easily performed, one right after the other, for rapid evaluation of the function of different enzymes.
Probing the biochemical modification of proteins.
For their study of the biochemical modification of heparin sulfate proteins, the scientists immobilized heparin sulfate proteins on magnetic nanoparticles. The fact that the nanoparticles are magnetic enabled the scientists to move the protein-coated nanoparticles to different regions on the chip (for the reasons discussed previously).
They first tested whether enzymes can biochemically modify proteins immobilized on the nanoparticles, in a conventional solution, for comparison to later experiments performed in the microfluidic device. The scientists utilized the enzyme D-glucosamino 3-O-sulfotransferase isoform-1, which is known to biochemically modify heparin sulfate proteins in the Golgi apparatus of cells, for this purpose.
The scientists found that the enzyme was able to biochemically modify approximately 5% of the proteins on the nanoparticles with sulfur-based chemical units. This number may seem low.
However, keep in mind that the proteins are densely affixed onto the nanoparticles. This means that only the protein segments at the periphery are available for chemical modification.
Also note that the enzyme the scientists utilized for this biochemical reaction is only able to chemically react with certain subunits of heparin sulfate proteins. Such specificity is a common functional feature of enzymes.
Functional consequences of the biological modification.
The scientists further found that the biochemically modified heparin sulfate proteins were able to chemically bind to an anticoagulant protein (antithrombin III), with 16 times the efficacy of unmodified heparin sulfate proteins. This demonstrates that the biological modification of the heparin sulfate proteins on the nanoparticles can enable important functional capabilities, as observed with heparin sulfate proteins in cells.
The highlight of this research was the scientists' demonstration of essentially the same results in their microfluidic device. They used a voltage to drive a drop of enzyme molecules and a drop of nanoparticles (coated with heparin sulfate proteins) into a mixing area.
They incubated the solution for 1 hour. This enabled biochemical modification of the heparin sulfate proteins on the nanoparticles with sulfur-based chemical units, as observed in the conventional solution experiments discussed previously.
Afterwards, the solution was taken from the microfluidic device, and the protein-coated nanoparticles were allowed to chemically bind to anticoagulant proteins. Enhanced binding was enhanced by a factor of 15, very similar to the results obtained in a conventional solution, demonstrating that biochemical reactions can be performed in the microfluidic device.
Further work with the microfluidic device may focus on investigating the specific biochemical modifications enabled by different enzymes on heparin sulfate proteins (or other proteins), rapidly and in parallel. Such knowledge is currently lacking for enzymes in the Golgi apparatus in living cells, but may be more readily obtainable from experiments performed with the microfluidic device described herein.
for more information:
Martin, J. G., Gupta, M., Xu, Y., Akella, S., Liu, J., Dordick, J. S., & Linhardt, R. J. (2009). Toward an Artificial Golgi: Redesigning the Biological Activities of Heparan Sulfate on a Digital Microfluidic Chip Journal of the American Chemical Society, 131 (31), 11041-11048 DOI: 10.1021/ja903038d