ISOLATION AND COMPOSITION OF NUCLEI AND NUCLEOLI 95 



The first and perhaps the most important limitation is that undoubtedly- 

 much low-molecular-weight material is extracted from nuclei during the 

 course of isolation, as well as presumably material of high molecular weight 

 such as protein. The degree of permeability of the nuclear membrane is at 

 the present time a matter of controversy, but in the face of a certain amount 

 of evidence in favor of permeability to material of as high molecular weight 

 as may be exhibited by protein, it is necessary to assume that considerable 

 loss of the latter may occur during the isolation of cell nuclei in aqueous 

 media, in addition to the loss of the unbound low-molecular-weight nuclear 

 constituents. The evidence against a high degree of permeability of the 

 nuclear membrane is at present meager. This subject will be considered in 

 greater detail subsequently, when the composition of nuclei with respect to 

 enzymes is discussed. 



The second limitation of major importance is the possibility of adsorp- 

 tion of cytoplasmic material during the isolation procedure, particularly 

 absorption of high-molecular-weight substances such as proteins. The ad- 

 sorption of soluble protein is not thus far known to constitute a serious 

 difficulty;^ but the adsorption of very finely divided particulate matter 

 may be serious in certain cases, as will be shown later on. Even if adsorp- 

 tion of a given soluble cytoplasmic protein does not occur on the nuclear 

 surface, there remains the possibiUty that this protein may form a com- 

 plex with deoxyribonucleic acid (DNA) and thus be bound by the nuclei. 

 Such a combintion will be caused to dissociate as a rule by the addition of 

 sodium chloride or by adjusting the pH to a sufficiently high value, since 

 nucleic acid and proteins generally form strong complexes only w^hen the 

 pH is above the isoelectric point of nucleic acid (which is very low) and 

 below or close to the isoelectric point of the protein in question.^ -^ 



In working with soluble proteins and enzymes, the investigator is there- 

 fore always in a dilemma since, if a high pH is used and if sodium chloride 

 is added, it is possible that nearly all of the soluble protein and enzymes 

 originally present within the nucleus may be lost. On the other hand, if the 

 pH is lowered so as to cause binding of intranuclear protein by allowing it 

 to form complexes vdth. DNA, an exchange of nuclear and cytoplasmic 

 protein may still occur to a sufficient extent to be troublesome.^ In addition 

 to this, the problem of adsorption of insoluble cytoplasmic material must 

 be considered, but such adsorption can be minimized if the nuclei are iso- 

 lated without concomitant rupture of cytoplasmic granules (mitochondria) 

 so that the latter can be removed from the nuclei while they are still intact. 

 This point \vi\\ be discussed in detail subsequently. 



1 A. L. Bounce and G. T. Beyer, J. Biol. Chem. 174, 859 (1948). 



2 E. Goldwasser and F. W. Putnam, J. Phys. and Colloid. Chem. 54, 79 (1950). 



3 L. G. Longsworth and D. A. Maclnnes, J. Gen. Phijsiol. 25, 507 (1942). 

 ^ A. L. Bounce, Expil. Cell Research Suppl. 2, 103 (1952). 



