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Abstract:
A new method of isolation and purification of the blue chromoprotein α-crustacyanin (prosthetic group = astaxanthin) from lobster shells is described. The amino acid composition was determined, and the equivalent weight was found to be 19,000 g chromoprotein per mole astaxanthin.
Under the influence of denaturing agents α-crustacyanin undergoes changes in quaternary structure accompanied by specific spectral changes. The molecular weights of α-crustacyanin and its subunits were determined by analytical ultracentrifugation, light scattering and osmometry (Table 3). α-Crustacyanin (λmax 632 nm) has a mol.wt. of 310,000–320,000; viscosity measurements ([η] ∼ 0.09 dl/g) indicate an anisometric molecule (axial ratio about 10:1).
In neutral solution of low ionic strength dissociation into eight subunits of mol.wt. 38,000 (β-crustacyanin, λmax 585 nm) takes place. α- and β-Crustacyanin are stable for months under proper conditions; stable products of mol.wt.between 40,000 and 300,000 could not be detected. In acid solution or in neutral solution containing 0.15% (w/v) sodium dodecysulfate, subunits of mol.wt. 19,000–20,000 (λmax 440 nm, 482 nm resp.) were found. In alkaline (pH 11.5–13) as well as in neutral solution containing urea (6–8 M), α- and β-crustacyanin dissociate further, yielding colorless protein subunits of mol.wt. about 7,000–10,000 (sedimentation constant 1.20 S), and high-molecular weight pigment aggregates (∼ 6 S), containing some protein (λmax of the total solution: 400 nm). Preparative separation of the pigment aggregates from the colorless protein subunits was effected by sucrose density gradient centrifugation in alkaline solution. In 1 N NaOH cleavage to still smaller fragments (s∼ 0.3–0.9 S) occurs, probably through hydrolysis of peptide linkages.
Extraction of the coloring matter from each of the products of different color (λmax 400 to 632 nm) by means of organic solvents, yields solutions showing the spectrum of unchanged astaxanthin.
The reversibility of the dissociation of α-crustacyanin in acid, alkaline (pH 12) and ureacontaining solutions is shown. On neutralizaton or dialysis the blue chromoprotein is regenerated, with λmax and sedimentation constant being the same as for native α-crustacyanin. However the formation of β-crustacyanin from α-crustacyanin solutions at low ionic strength is irreversible. The subunits of mol.wt. 20,000 and 7,000–10,000, resp., can be obtained starting from β-crustacyanin instead of α-crustacyanin, but neutralization or dialysis then yields only β-crustacyanin, with no α-crustacyanin.
In some instances the denaturation was observed kinetically on the basis of color changes. The denaturation of α-crustacyanin in extremely concentrated electrolyte solutions was investigated in the same way; the extent of denaturation was found to depend mainly on the electrolyte concentration and less on specific properties of the investigated salts.
The relation between the color changes and quaternary protein structure as a function of the degree of solvation of the astaxanthin molecule and protein is discussed. Presumably in α- and β-crustacyanin the astaxanthin is enclosed by protein, while in the smaller subunits it is partially exposed to water. Solvation of astaxanthin by water is very poor; dilution of a concentrated dimethylsulfoxide solution of astaxanthin by water leads to a shift in λmax to about 440 nm. The shift to 400 nm or 440 nm, which is observed during the dissociation of crustacyanin in the presence of urea or alkali or acid resp., is interpreted as a similar solvation effect.
