At first glance, Dr. Ali Zand's research on the chemistry of color vision defies the very foundation of the art world, where red, blue and yellow are the "primary colors" from whence all other colors are made. In the chemistry world, the colors are determined by red, blue and green proteins- heresy to those schooled in the universally accepted theory of the color wheel.

According to Zand, associate professor of Chemistry at Kettering University, the human eye perceives all colors based on three colored proteins (rhodopsins): red, blue and green. Before all the artists and art majors start rioting in the streets, take a moment to consider the chemistry that backs up Zand's claim.

"Why humans see different colors is based on how these proteins react in the cone cells of the eye," he said. "There is only one chromophore (one molecule) that is responsible for a chemical reaction that takes place in the eye allowing humans to see," Zand said, "that molecule is Retinal, a form of vitamin A."

Retinal combines with a protein called Opsin to form Rhodopsin, the chemical entity (protein) responsible for vision. Opsins are found in the rod and cone cells of the eye. Zand's research is concerned with the question 'If there is only one molecule involved in vision, how can that one molecule allow us to see so many different colors?"

To find out, Zand, and Dr. Babak Borhan, associate professor of Chemistry at Michigan State University and his research group, collaborated to engineer a protein that could mimic Rhodopsin's response to chemical and physical changes. They had to engineer a surrogate protein because Rhodopsin is a membrane-bound protein that could not easily be separated and purified for research purposes.

"Rhodopsin has to stay within the membrane to maintain its conformation (shape)," said Zand. "We engineered a protein called Cellular Retinoic Acid-binding Protein II (CRAP II - who said scientists don't have a sense of humor?) to use in research."

To understand the importance of this CRAP II protein, it's necessary to explain the process of vision. When the light hits the eye it passes through the cornea, the lens and the vitreous fluid. Those three objects focus the light on the tissue lining the inner part of the eye, which is the retina. The retina is made up of thousands of rod and cone cells that are activated through the absorption of light by Rhodopsin.

Inside of the active site of Opsin is 11-cis-Retinal. The chemical structure of cis-Retinal has a bend in its carbon structure that straightens out (becomes more planar) when light is absorbed. This causes a cascade of events. Rhodopsin is a G-protein coupled receptor; it binds itself to and activates the G-protein called transducin. When Rhodopsin absorbs light, transducin releases a subunit of its molecular structure called the alpha segment. The alpha segment attaches itself to another protein leading to cascade of events resulting in closure of calcium ion channels within the cell. This causes a gradient in ion concentrations inside the cell versus outside the cell, initiating an electrical impulse that leads to a neurotransmitter release into a synapse. The neurotransmitter is picked up by another neuron and the neuron transmits the signal to the brain resulting in a visual image.

'The rod cells are very sensitive to different shades of light and darkness; and cone cells are actually the cells that allow humans to see color', said Zand.

The rod cell has an outer segment that has various membranous discs containing a seven-alpha-helical protein (seven alpha helixes that are bound together like a barrel) called Opsin. Cone cells possess the same discs, but they are part of the actual cell membrane of the cone cell, not disconnected from the plasma membrane as in the rod cells.

In the cone cell there are three different types of Opsins: Red Opsin, Blue Opsin and Green Opsin. The wavelength where Retinal absorbs light is different for each. The Blue Rhodopsin absorbs at 420 nanometers; Green Rhodopsin absorbs at 530 nanometers and Red Rhodopsin absorbs at 570 nanometers.

There may be only one chromophore (Retinal), but because the proteins are a little different, the way the Retinal is attached to a protein has a great deal of affect on its wavelength of absorption.

"Color blindness results when the absorption wavelengths of Red Rhodopsin and Green Rhodopsin are closer together. Red and Green Rhodopsin are 96 percent sequence identical," said Zand. All the rhodopsins have about 348 amino acids and two oligosaccharide (sugar) chains. Human red and green cone proteins differ by only 15 amino acids.

Hence, the color blindness arises from either mutations or lack of one or more of the opsin proteins. Although color vision deficiencies are termed color blindness, in reality it is simply a reduced ability to distinguish between colors. Most people who are color blind cannot distinguish between red and green because mutations in Red Rhodopsin can lead to shift in absorbance from 570 nm to 530 nm and vice versa. These individuals can, however, distinguish between blue and red or blue and green because blue absorbs at a much lower wavelength. However, they cannot distinguish between red and green.

"There are people who lack cone pigments or within whim the Opsins within the cone cells are non-functional, and they can only see shades of black, white and gray," Zand said, "but it's very rare."

Scientists have proposed many different theories to explain why this one molecule absorbs light at three different wavelengths and allows us to see different colors.

One theory is related to how the protein arranges itself around the Retinal and the bend in the carbon structure of Retinal. When the structure is bent, the carbons are not on the same plane. The more bent the structure the more blue-shifted the light absorption is going to be.

Another hypothesis relates to the point at which Retinal attaches to Opsin. At the attachment point there is a nitrogen molecule with a positive charge. Because the positive charge requires a counter ion- a negative charge, the location of that negative charge is very crucial. The Retinal becomes more planar in the active site as the distance between the positive and negative charges increases. Therefore, it is hypothesized that the negative charge and positive charge are very close in Blue Rhodopsin whereas they are far apart in the Red Rhodopsin.

Zand and the researchers at Michigan State University have engineered the CRAP II protein to bind to Retinal instead of Retinoic Acid, and they are currently looking at whether they can prove these hypotheses by measuring how twisted the molecule is within the active site as well as trying to mimic the Rhodopsin in changing the positions of the negative charges. Their tests will attempt to show if a shift in the absorption from red to green to blue can be accomplished by merely changing the position of the counter-ion (negative charge) within the protein active-site, or by causing different twists in the Retinal.

Could unraveling the chemistry underlying our color vision enable science to chemically alter someone's eye so they could see color if they were colorblind? "The only way that could be done is through stem cell research or gene therapy, by inoculating the eye with stem cells that would eventually differentiate into rod and cone cells possessing healthy proteins enabling the person to see color," said Zand.

For all those artists who still insist the primary colors are red, blue and YELLOW, Zand sympathizes, but said yellow is merely "green" with envy.

Written by Dawn Hibbard
(810) 762-9865
dhibbard@kettering.edu