The Oxidation of Hemocyanin

Hemocyanin is a copper-containing protein which occurs in the blood of a number of species of arthropods and molusks. The extent of the reversible combination with molecular oxygen is a function of the partial pressure of oxygen, and like the formation of oxyhemoglobin is affected by changes in acidity and salt concentration. The ratio of combined molecular oxygen to copper is 02:2Cu in all the hemocyanins studied from a variety of different bloods. The deoxygenated protein is colorless, while the oxygenated compound (oxyhemocyanin) has an int’ense blue color. No evidence has hitherto been available as to whether the copper in hemocyanin was in the cupric or cuprous state. The usual oxidizing agents which oxidize the ferrous compounds, hemoglobin and oxyhemoglobin, to the ferric compound, methemoglobin, appear to be without effect on hemocyanin or oxyhemocyanin. We have now found that by the use of the two very powerful oxidizing agents, potassium molybdicyanide or potassium permanganate, it is possible t’o oxidize the hemocyanin (or oxyhemocyanin) of Limulus polyphemus. In this way two new proteins are formed in which the copper is in the cupric state. One of these, prepared from hemocyanin in the absence of oxygen, is colorless and we shall designate it as methemocyanin. The other, oxymethemocyanin, is formed when a solution of methemocyanin is shaken with air or oxygen; the deoxygenation of methemocyanin like that of hemocyanin may be brought about by diminishing the partial pressure of the oxygen above the solution. It is evident that, unlike methemoglobin, methemocyanin combines reversibly with oxygen. The cupric compounds, methemocyanin and oxymethemocyanin, are reduced by the action of a variety of reducing agents,