THE INDIVIDUAL GROUPS OF PROTEINS 491 



CRYSTALS OF OXYH^EMOGLOBIN AND DERIVATIVES. 



The ease with which crystals of oxyhsemoglobin can be prepared fiom 

 the blood of animals differs very considerably and numerous methods have 

 been tried. Generally, only sufficient crystals lor microscopic examination can 

 be prepared ; the preparation is difficult and frequently unsuccessful. Larger 

 quantities of crystals can be prepared from the blood of the horse and of 

 the ox. 



Oxyhsemoglobin, 



Preparation for Microscopic Examination. 



(a) From rat's or horse's blood. An equal volume of distilled water 

 and i or 2 drops of ether are added to the blood and mixed thoroughly. A 

 drop of the solution is placed on a microscope slide and covered with a cover 

 slip when the edges begin to dry. Crystals gradually separate out. 



(b) From dog's, rat's, guinea-pig's, or horse's blood. The defibrinated blood 

 is laked by adding ether, or better ethyl acetate, drop by drop. Ammonium 

 oxalate solution is added until 1-5 per cent, is present. Crystals of 

 oxyhaemoglobin separate out, especially if the solution be cooled to o. 



(c} From dog's, rat's, squirrel's, man's, rabbit's, or pig's blood. The 

 defibrinated blood is laked by freezing and thawing several times in a platinum 

 basin. After the last thawing it is poured into a dish in layers less than i '5 cm. 

 thick. Crystals separate out if the dish is set aside in a cool place. 



(d] From dog's, ox, rabbit's and other bloods. One-sixteenth of its volume 

 of ether is added to the defibrinated blood and the blood is laked by shaking 

 for some minutes in a stoppered bottle, the stopper being removed to allow 

 air to escape as the ether evaporates and being allowed to become coated with 

 dry blood so as to prevent the entry of air. The bottle is kept at an even tem- 

 perature for two or more days. On removing a drop, placing it on a microscope 

 slide, covering it when the edges commence to dry, crystals gradually form. The 

 crystals are crystals of haemoglobin. 



If human blood be mixed with putrid sheep's serum it will give crystals 

 by this method (Copeman). 



Preparation in Large Quantities. 



Ox blood or horse's blood is defibrinated and strained through muslin and 

 centrifuged at 2000-2400 revolutions per minute. The serum is decanted, 

 the sediment of corpuscles stirred up with 0*85 per cent, sodium chloride 

 in amount equal to that of the serum and again centrifuged. The solution 

 is decanted and the sediment dissolved in the smallest quantity of water at 

 37. To the solution cooled to o is added half its volume of ice-cold 

 ether; the mixture is placed in a separating funnel, shaken several times 

 and allowed to stand for 24 hours at o. Of the three layers which form, the 

 lowest clear one is carefully collected (the middle one contains residues of 

 corpuscles and is gelatinous, the top one is ether), filtered into a flask kept at 

 o and freed from ether by a current of air purified by passage through per- 

 manganate and sulphuric acid. Ice-cold alcohol is added to the ice-cold 

 solution one-third of the volume in the case of ox blood, one-quarter in 

 the case of horse's blood. This mixture is kept in a freezing mixture. In 12 

 hours crystals are obtained from horse's blood, in 24 hours from ox blood. The 

 crystals are separated by a centrifuge, if possible at o, washed with^ ice-cold 

 20 per cent, alcohol and then water, dissolved in distilled water at 30, cooled 

 to - 3 and left to crystallise. This is repeated 2 or 3 times. The crystals are 

 finally placed on filter paper and dried in vacuo for 6 hours over sulphuric 

 acid. They are powdered and dried in hydrogen at 100. 



Crystals of oxyhasmoglobin may be more rapidly prepared from horde's 

 blood by Abderrulden's modification of the Zinoffsky method: 



