1912.] Studies on the Reductase of Liver and Kidney. 487 



II. Technique. 



As previous experience had shown the high probability of the enzyme being 

 comparatively insoluble, we decided in some instances to perfuse the liver or 

 kidney, as the case might be, with tap-water rather than with physiological 

 saline. We desired not to have to deal with a saline press juice, because it 

 was hoped that we might be able to employ the method of following changes 

 in electrical conductivity as quantitative in investigating the progress of 

 reduction. Our hopes in this connection were not realised, so that the liver 

 used on March 18, 1912, was perfused with physiological saline. 



The preparation of the juice on March 27 may be taken as typical. We 

 were supplied with the half of an ox liver from an animal which had just been 

 killed, and this we perfused with tap-water heated to 40° C. until the 

 emergent water was bloodless. The liver was then cut into large pieces, and 

 a good deal of the water allowed to flow out of it. The pieces were then cut up 

 into much smaller portions and forced into the juice-press, in which they were 

 crushed under very considerable pressure. A fawn coloured, viscid liquid 

 dripped out and was received under toluene. This juice was subsequently 

 filtered through cheese-cloth to free it of connective tissue and the debris of 

 blood-vessels. Juices were similarly prepared on January 16 from a frozen 

 ox-kidney, on February 5 from perfectly fresh pig's liver, and on March 18 

 i'rom pig's liver. 



III. Experimental. 



(a) Observations with Methcernoglobin. — We were very anxious to make 

 observations on the action of the juice on haemoglobin. It was not possible 

 at the time of our experiments to get a sufficient quantity of fresh mammalian 

 haemoglobin, so that we used dried " haemoglobin scales, soluble, Merck." 

 On dissolving some of these dried scales, and filtering the solution, we found 

 that the pigment gave the spectrum of methaemoglobin, that is, had the band 

 in the red along with the two bands of . oxyhemoglobin. The solution was 

 stable, and was unaltered by bubbling air (freed from C0 2 ) through it for 

 four hours ; the colour was brown, and never became crimson, so that it was 

 still methaemoglobin. Two equal volumes of the brown solution were now 

 taken, and to one of them 6 c.c. of ox-liver juice, two days old, were added, 

 and to the other 6 c.c. of the same juice heated for 10 minutes at 100° C. 

 The juice and the methaemoglobin were shaken together and placed in a 

 thermostat at 40° C. In less than 10 minutes the unboiled tube was of a 

 bright pink colour, the boiled control being of the original rusty brown. 

 On filtering off, the clear, pink liquid from the opaque juice, and examining it 

 with the spectroscope, the two bands of oxyhemoglobin were very distinct, 



2 M 2 



