Katelyn Boone

Defense Date


Graduation Date



Immediate Access

Submission Type


Degree Name



Environmental Science and Management (ESM)


Bayer School of Natural and Environmental Sciences

Committee Chair

Brady A. Porter

Committee Member

John F. Stolz

Committee Member

Kyle W. Selcer


Biliverdin, Blue pigment, Chromoprotein, Coloration, Etheostoma blennioides, Etheostoma caeruleum


Unlike most other vertebrates, many species in the genus Etheostoma do not utilize structural refraction to display blue or green color. Instead, blue and green mating coloration exhibited by male rainbow darters (E. caeruleum) and male greenside darters (E. blennioides) results from the presence of true chromoprotein pigments. This study was conducted in order to extract, purify, characterize, and compare these novel pigments. Pigments were extracted in aqueous buffer and partially purified by ammonium sulfate fractionation and gel filtration chromatography. Final purification consisted of preparative non-denaturing polyacrylamide gel electrophoresis for E. caeruleum and hydroxyapatite chromatography for E. blennioides. Isolation of the chromophore was accomplished using acetone precipitation. The chromophore is the same in both species and is believed to be biliverdin. The protein component differs between the species and appears to have a greater number of subunits in E. blennioides. Binding of the protein to the chromophore amplifies the absorbance in the visible region and causes spectral tuning of the absorbance profile of the chromophore, with slight differences between species. In E. caeruleum, the chromoprotein pigment has a lambda max of 683 nm and transmits light at slightly shorter wavelengths, causing it to appear blue. In E. blennioides, the chromoprotein pigment has a lambda max of 696 nm and transmits light at slightly longer wavelengths, causing it to appear green. This work has shown that the protein component, not the chromophore, is responsible for the difference in hue between these two pigments. Future work will involve obtaining amino acid sequences for the protein component of the pigments and ultimately sequencing the gene coding for these proteins in darters.