A hierarchy of disulfide-bonded subunits: the quaternary structure of **Eudistylia** chlorocruorin

The quaternary structure of the cysteine-rich, ∼3500-kDa chlorocruorin (Chl) from the marine polychaete Eudistylia vancouverii was investigated using maximum entropy deconvolution of the electrospray ionization mass spectra (ESIMS). The native Chl provided two groups of peaks, at ∼25 and ∼33 kDa, and one peak at ∼66 kDa. ESIMS of the reduced and reduced and carbamidomethylated Chl and of its subunits obtained by HPLC provided the complete subunit composition of the Chl. Two groups of nonglobin linker chains were observed:  L1a−f (25 000.4, 25 017.9, 25 039.6, 25 057.0, 25 074.4 and 25 096.8 Da) and L2a−d (25 402.7, 25 446.0, 25 461.6 and 25 478.3Da) (±2.5 Da), with relative intensities L1:L2 = 5:2. Six globin chains were found, a1, a2, and b1−4, with reduced masses of 16 051.5, 16 172.4, 16 853.5, 17 088.9, 17 161.2 and 17 103.6 (±1.0 Da) and relative intensities of 8:4:1:4:2:1, respectively. Disulfide-bonded dimers and a tetramer of globin chains were identified:  D1 = a1 + b3 at 33 207.1; D2 at 33 374.1...