Size-exclusion high performance liquid chromatography of the dodecameric and hexameric forms of hemocyanin from Callinectes sapidus

The relationship between the polymeric composition of hemocyanin and environmental conditions could be described more thoroughly if there were a simple method for separating the dodecameric and hexameric forms of the protein. Such a method for the separation of these two forms of hemocyanin from Callinectes sapidus Rathbun using size-exclusion high performance liquid chromatography has been developed. The method uses columns with an exclusion limit of 1x10⁶ daltons and monitors the binuclear copper moiety by its absorbance at 337 nm. The application of the method was demonstrated by obtaining the dodecamer to hexamer ratios for 20 hemocyanin samples from freshly caught adult intermolt male C. sapidus. The ratios ranged from 1.9:1 to 7.3:1 and appeared to be normally distributed. This variation in the polymeric composition of hemocyanin (much greater than that attributable to the reproducibility of the assay) is apparently occurring in nature, contrary to previous reports that have indicated the ratio to lie within a narrow band for a given species.New address effective May 1, 1992: Department of Chemistry, University of California, Irvine, California 92717, USA