BOLTON, HOYER, AND RITTER 



19 



sure cell 2 operated at approximately 10,000 psi. Break- 

 age of the bacteria by this means is essentially com- 

 plete. The resulting bacterial juices were examined in 

 the analytical ultracentrifuge (Spinco, Model E) as 

 soon as practicable (about 30 minutes after rupture), 

 or after various periods of storage at 4° C. The 

 centrifuge was routinely brought up to speed in 6 to 

 7 minutes and held at about 60,000 rpm for the dura- 

 tion of the run. 



RESULTS 



Figures 1 to 4 are illustrative sedimentation dia- 

 grams showing that the pattern of rapidly sediment- 

 ing components varies in accord with the kind of 

 suspending medium used. Figure 1 compares the 

 sedimentation behavior of the components in extracts 

 prepared from bacteria broken in 0.01 M Tris-0.004 M 

 succinic acid-0.005 M magnesium acetate buffer (pH. 

 7.6, "TSM"), in 0.01 M Tris-0.004 M succinic acid 

 ("TS"), or in TSM + 0.07 M phosphate (pH 7.6). 

 Values spotted along the abscissa are approximate ap- 

 parent sedimentation coefficients. It is evident from 

 this comparison that more, and larger, components 

 are observed when magnesium has been included in 

 the buffer and also that phosphate abolishes the more 

 rapidly sedimenting materials. Whether the effect of 

 phosphate is specific or whether the result is due to an 

 increased ionic strength of the medium is not known. 

 The sharp spike characteristic of highly polymerized 

 deoxynucleic acid (DNA) 3 is missing from these dia- 

 grams, although it is readily observed in juices pre- 

 pared by breaking E. coli as a result of lysozyme treat- 

 ment and osmotic shock. In spite of this finding, 

 three-quarters of the ultraviolet-absorbing substance 

 and one-seventh of the protein of E. coli disrupted in 

 the TSM medium may be sedimented in the prepara- 

 tive rotor (100,000g, 90 minutes). 



Figure 2 shows that juices prepared by pressure 

 cell disruption maintain constant sedimentation dia- 

 grams for at least 20 hours. If, however, sodium 

 ethylenediaminetetraacetate (EDTA, 0.1 M, pH. 7.6) is 

 extracts, all components greater than about 20 S disappea 



TSM + 

 07M PO, 



iltaW 



8 mm 60,000 RPM 



Fig. 1. Sedimentation 

 diagrams of E. coli dis- 

 rupted in various buffer 

 solutions. The concentra- 

 tion of the bacterial juices 

 differed among the runs. 



added to the bacterial 

 r. This occurs whether 



2 C. S. French and H. W. Milner, Methods in Enzymology I, Academic Press, p. 65. A 

 similar device is marketed by the American Instrument Company, Silver Spring, Maryland. 



3 See, for example, the sedimentation diagrams reported by W. Gillchriest and R. Bock, 

 S. Dagley and J. Sykes, and J. Wagman reported in the present volume. 



