BIOCHEMICAL STUDIES OF PHYLOGENY 115 



texts on biochemistry (Harper 1959; Baldwin 1957; Fruton and 

 Simmons 1958). 



A thorough survey of the distribution of phosphagens CP and 

 AP in the animal kingdom was published in 1931 by Needham, 

 Needham, Baldwin and Yudkin. In some cases they dissected 

 out the muscle tissue from the lower animals ; in other cases they 

 used the whole animal, the method used depending upon the 

 size and availability of the raw material. 



It should be remembered in all the discussions of their experi- 

 mental work that most of the workers were pioneers in the field 

 and that present-day criticism of techniques is in no way meant to 

 be disparaging. It is only too easy to look back over a quarter of a 

 century of research and, being wise after the event, to point out 

 the various faults and errors. It is inevitable in a scientific subject 

 that the years will bring great improvements in techniques which 

 will then indicate that the previously used methods and con- 

 clusions were not sufficiently justified. There is but one way of 

 making sure that one's work will never contain any errors and 

 that is to do no work. 



The technique that Needham et al. used for their analysis of the 

 phosphagen was as follows. They cooled their material and dis- 

 sected out the required part. This was then weighed, ground up 

 with trichloracetic acid, left for 10 min in the cold and then 

 filtered. The filtrate was neutralised with NaOH and then CaCl 2 

 was added to precipitate the inorganic phosphate. This pre- 

 cipitate of insoluble calcium phosphate was spun down in a 

 centrifuge and separated from the supernatant fluid. The 

 precipitate was dissolved in a few drops of concentrated sulphuric 

 acid and the inorganic phosphate then determined. 



The organic phosphate was still in the supernatant solution and 

 it might contain the two possible phosphagens, creatine phosphate 

 and arginine phosphate (CP and AP). These were analysed as 

 follows. If one places CP (or AP) in acid solution, it hydrolyses 

 to form either creatine (or arginine) and phosphoric acid. If 

 molybdate ions are present the CP hydrolyses much more rapidly 

 than does AP. Thus the determination of phosphate after 15 min 

 hydrolysis gave an indication of the CP value whilst estimation 

 after 15 hr gave both CP and AP values. The value of AP could 

 then be determined by subtraction. 



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