The Specific Precipitin Reaction of the Muscle Hemoglobin of the Dog

Preparation of Muscle Hemoglobin.-The method of preparing muscle hemoglobin may be described as follows: The animal is freed from blood by the technic used in other experiments to wash out plasma proteins. Under ether anesthesia cannulas are tied into the carotid and jugular vessels. A Ringer's solution with 5% glucose at body temperature is injected by gravity into the jugular vein at the same speed as blood is drawn from the carotid. By slow and careful simultaneous bleeding and transfusion one can reduce the red cell hematocrit from 50% (or normal) to very low values of '2 to 3%. When the heart stops the thorax is opened immediately, a cannula placed in the first part of the aorta, the right auricle incised and gravity perfusion continued freely at a pressure of about 100 cm. of solution for a few minutes, utilizing 3 to 4 liters of perfusate. The two instances in which the muscle hemoglobin solutions gave slight precipitin reactions for dog blood hemoglobin were experiments in which this gravity perfusion was omitted. The procedure obviously washes out the last traces of circulating blood. Massage of trunk, back, thorax and extremities is continued during the last perfusion period. Then the thoracic and abdominal viscera are rapidly removed and further massage of the muscles is continued to remove any traces of perfusate. The various muscles are then dissected free with no loss of time, rinsed and massaged in physiologic salt solution. The tissue is then dried with a towel and care is taken to remove any gross fat or fascia. The bloodfree muscles are ground in a meat grinder and the tissue diluted with four volumes by weight of distilled water or 0.4% ammonia solution. The ammonia solution gives more complete extraction of the hemoglobin from the muscle. The mixture is thoroughly stirred and placed in an icebox (2 to 4 C.) over night. This red solution is now strained through cloth and cleared in large part by centrifugalization which also remove troublesome fat, then filtered through paper. The solution may then be passed through a coarse Berkefeld filter and finally through a finer one to remove any bacteria remaining. This last procedure removes a good deal of muscle hemoglobin but is necessary for certain experiments. The solutions of muscle hemoglobin used in these experiments contained from 600 to 800 mgm. muscle hemoglobin per 100 gmin. muscle. The sterile clear red solution was then shipped in sealed containers by mail for the precipitin tests to be described. A general description of the various methods used for analysis is given in a recent paper.'