E. HOFF-J0RGENSEN 



deoxynucleoside present. The deoxynucleoside content of a sample is determined 

 by interpolating the response to the known amount of the test solution onto this 

 standard curve. The deoxynucleoside content per ml. of the test solution is now 

 calculated for each of the duplicate sets of tubes, and the deoxynucleoside content 

 of the sample is calculated from the average of the values. 



Preparation of samples for assay 



Deoxynucleosides : a solution containing about 3 m/zmol. (or 05-10 /xg.) 

 deoxynucleoside per ml. is prepared in water, or in a not-more- than o 05 m maleic 

 acid buffer at pH 67. 



Deoxynucleotides : incubation of a solution of deoxynucleotides with crude intes- 

 tinal phosphatase (Schmidt and Thannhauser, 1943) is without effect on the response; 

 it is therefore concluded that deoxynucleotides give the same response as deoxy- 

 nucleosides on a molar basis. 



Deoxynucleic acid. Pure DNA has a growth effect which is less than 1 per cent, 

 of the effect of the deoxynucleosides present in the DNA. If, however, the DNA is 

 depolymerized by deoxyribonuclease, (Kunitz, 1950) the growth response is equival- 

 ent to the effect of the calculated content of deoxyribosides in the DNA. Samples of 

 bacteria, yeast or tissue may either be analysed in the wet state or after drying with 

 acetone. For the analysis of bacteria and yeast, the cells should be disintegrated, e.g. 

 in 'the tuning-fork disintegrator' (obtained from H. Mickle, Hampton, Middlesex, 

 England). The sample containing at least 0-2 fxg. P as DNAP is placed in a small test- 

 tube. An exactly measured amount of 0-5 n NaOH solution (e.g. 0-5 ml.) is added, or 

 if the sample is a solution, enough 1 -o n NaOH solution to make the final solution 

 05 n in NaOH. The tube is placed in a boiling water-bath for 15 min. During this 

 time the tissue is disintegrated with a glass rod. After the incubation at ioo° C. 5 vol. 

 of a solution containing 0-06 g. mol. of maleic acid and o-oi g. mol. of magnesium 

 sulphate per 1. are added for each vol. of 0*5 n sodium hydroxide used above. The pH 

 of the mixture should now be 6-3-7-0. In order to depolymerize the DNA 01 ml. 

 of a solution usually containing 100 /xg. of crystalline deoxynuclease (Worthington, 

 Biochemical Lab., Freehold, New Jersey, U.S.A.) is added and the mixture is incub- 

 ated for 16-20 hr. at 37 G. For each new material assayed, the minimum amount 

 of DNAase which gives maximum response should, however, be found by experi- 

 ments. After incubation the mixture is diluted to contain about 3 mju. mol. deoxy- 

 nucleoside per ml. and assayed (one g. mol. deoxynucleoside — ' 310 g. DMA). 



Differentiation between purine and pyrimidine deoxynucleosides 



As the pyrimidine deoxynucleosides are stable towards mild acid hydrolysis, 

 whereas the purine deoxynucleosides are not, it is possible to distinguish between these 

 two types of deoxynucleosides by assaying the depolymerized sample before and 

 after boiling for 5 min. at pH 1 . Before assaying the acid solution must be neutralized. 



Specificity, sensitivity and accuracy 



The method seems to be absolutely specific for the deoxyribonucleic linkage and 

 allows the determinations of amounts greater than about 2 jug. of deoxynucleosides, 

 deoxynucleotides or DNA with a standard deviation of about 5 per cent. 



82 



