Functional properties of chemically modified hemocyanin. Fixation of hemocyanin in the low and the high oxygen affinity state by reaction with a bifunctional imido ester.
Hemocyanin of Helix pomatia is a respiratory protein with a molecular weight of 9 times 10-6; it contains 180 oxygen binding sites. The reaction of hemocyanin with the bifunctional reagent dimethyl suberimido ester, which reacts with amino groups, has been studied. Up to 75 per cent of the amino groups can be modified without inactivation of oxygen binding sites or dissociation of the protein, It appears that hemocyanin can be fixed in a state with low oxygen affinity by modification of the deoxy protein, and in a state with high oxygen affinity by modification of the oxy protein. Using conditions under which native hemocyanin binds oxygen cooperatively (Hill coefficient 2.9), modification of deoxy- and oxyhemocyanin yields derivatives with different oxygen affinities (P50 equals 10  and 2.2 mm, respectively). Both the deoxy and oxy derivatives show strongly reduced cooperativity (Hill coefficients 1.4 and 1.1, respectively). Modification of oxy- and deoxyhemocyanin subunits (molecular weight one-tenth of the native protein), which bind oxygen noncooperatively, results in derivatives with oxygen binding properties identical with those of unmodified subunits. Parallel experiments have been carried out with a unifunctional reagent, methyl acetoimido ester. Modification of partially oxygenated hemocyanin under conditions at which the protein binds oxygen cooperatively yeilds derivatives with redued cooperativity (Hill coefficents 1.1-1.2) and an oxygen affinity depending on the oxygen saturation whivh modification had been carried out. The results are consistent with a simple two-state model for the cooperativity of oxygen binding by these giant hemocyanin molecules.
D000818:Animals; D001665:Binding Sites; D002118:Calcium; D003998:Dicarboxylic Acids; D006372:Helix, Snails; D006433:Hemocyanins; D007094:Imides; D007700:Kinetics; D010100:Oxygen; D010313:Partial Pressure; D011485:Protein Binding
D003998:Dicarboxylic Acids; D007094:Imides; D006433:Hemocyanins; D010100:Oxygen; D002118:Calcium