Diffusion coefficient subunits

For the purpose of this calculation it was necessary to know the diffusion coefficient for ligand and whole molecules of hemocyanin (Table I) and the value of the diffusion coefficient for half molecules of hemocyanin. If the level of calcium ion is reduced in pH 9.6 glycine buffer to the point where no whole molecules remain, the half molecules always are contaminated with one-twelfth size (5S) subunits, so that it is impossible to measure directly the diffusion coefficient of half molecules. For a self-associating system, the Gouy method leads to an average diffusion coefficient which is the Z-average, defined by... 

Many physical characteristics have been determined for the mushroom tyrosinases. These include diffusion coefficients, sedimentation coefficients, frictional ratios, Stoke s radii, isoelectric points, and apparent molecular weights (17-20). Recent evidence suggests that tyrosinase is composed of two heavy chain components (H) of approximately 43-45 kd each and two light chain components (L) of approximately 13 kd each (18-20). The heavy chains contain the catalytic sites. Podila and Flurkey (21) have suggested that the heavy chains were synthesized as smaller molecular weight precursors to the native enzyme. Different isoenzyme forms of tyrosinase apparently contain different H chains (7,17). No role for the L chain subunits have been reported. 

The P subunit sedimented as a single sharp band with S20fW= 3.54 +/- 1.9% (not shown). Using the diffusion coefficient and partial specific volume calculated for R.rubrum, a molecular weight of 45,000 was calculated which suggests that the P subunit (molecular weight 54,000) exists as a monomer in solution. This is supported by the fact that P coeluted with BSA (molecular weight 66,700) after gel filtration. 

The molecular weight of the monomeric form of the enzyme was approximately 480,000, as calculated from sedimentation and diffusion coefficients. Dissociation in guanidine hydrochloride yielded subunits of mol. w. 69,100, suggesting the presence of 7 subunits per molecule. However, there was some uncertainty as to the true value for the partial specific volume, and the molecular weight of the subunits could actually be about 60,000. This value would correspond to 8 subunits per molecule, and electron micrographs of the crystalline enzyme rather lent support to an octomer model. 

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