Abstract
The object of this communication is to report on a new observation that tetanus toxin (tetanospasmin) may be found intimately associated with the hemoglobin of guinea pigs during the paralytic stages of the disease. A part of an extensive series of experiments on this problem is briefly summarized, as follows:
Guinea pigs weighing each 350 g were injected subcutaneously in the abdominal region with tetanus toxin∗ in doses ranging between 20 to 40 M.L.D. and bled from the heart during the stages of complete paralysis and spasmodic contractions (i.e., 24-48 hours following the subcutaneous injection). The pooled blood was defibrinated by shaking with glass beads; filtered through several layers of gauze, and centrifuged at room temperature for one-half hour. The clear, dark red supernatant plasma and the sedimented red blood cells were placed in separate containers.
Washing of red blood cells in 1% NaCl solution was repeated until the supernatant fluid became biuret-negative (usually 4 washings are required). The washed cells were diluted 1:5 in distilled water. When fairly complete laking and some crystallization occurred about one hour later, the solution was centrifuged for one-half hour also at room temperature. This procedure resulted in separation of a dense dark red sediment containing masses of hemoglobin crystals; a superimposed loose whitish precipitate consisting of cellular debris and some hemoglobin crystals; and finally, a clear dark red supernatant fluid.
The serum was fractionated with the aid of the somewhat modified method of Parsons, 1 originally recommended for crystallization of hemoglobins and in these experiments consisting of repeated alternate freezing to -14°C and centrifugalization of the serum at 4°C until complete thawing. The procedure enabled the separation of colorless, faintly colored, medium colored, and dark red fractions. Small amounts of crystals and whitish debris were also obtained.
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