Chapter 11 



STUDIES OF PROTEOLYTIC EXZY^IES IX BOXE .MARROW 



By WILLIAM G. BATT and CHARLES A. ZITTLE 



The object of this investigation was to study the presence of any pro- 

 teolytic enzymes that may be present in bone marrow. If the evidence 

 warranted it, a comparison was to be made between the proteolytic enzyme 

 content of non-irradiated animals and animals which had been subjected 

 to the influence of neutron radiation. 



A preliminary study was made to determine the optimum pH range 

 and the limiting concentration of substrate. The technic employed fol- 

 lowed that developed by Linderstr0m-Lang and Holter (1) and Weil and 

 Jennings (2), but crystallized bovine plasma albumin was used as the 

 proteinase substrate for the standardization experiments. 



The results obtained when using minced rabbit kidney extracted with 

 60 per cent glycerol agreed with Weil's value for the optimum pH (7.5) 

 but differed slightly in the limiting concentration of the substrate. It was 

 found that the plateau began nearer 8 per cent rather than the lower value 

 of 5 per cent. 



After establishing these conditions, a study was begun to detect the 

 presence of proteolytic enzyme in bone marrow. X'o attempt was made 

 to separate the red and yellow portions, but a pooling of all the marrow 

 was used. This was divided, however, into 2 portions, one being extracted 

 with 60 per cent glycerol and the other with physiological saline solution. 

 Xo appreciable difference was noted on comparing the values obtained 

 from both extracts. In both cases they were negligible. Changes in 

 concentration of bone marrow and increase in the time of reaction gave 

 very little increased value. Changing the temperature from 25° to 37° 

 was of no advantage. 



With no apparent reaction between the bone marrow extract and bovine 

 plasma albumin it was decided to change the substrate to d-1 leucylglycyl- 

 glycine. The results were similar to those obtained with the previous 

 substrate. 



In order to check the efficiency of extraction of the bone marrow a sample 

 was obtained which had been sonicized by submitting it to intense vibra- 

 tions of 10.5 kc. provided by a Pierce magnetostriction oscillator of the 

 Chambers and Flosdorf type (3). This sample was incubated with d-1 

 leucylglycyl-glycine at 37°C. but no appreciable difference was obtained 

 as compared with the non-sonicized ones. 



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