Nucleoprotein solutions

Four processes are concerned in the isolation of a nucleic acid. First is the destruction of the tissue structure (stage 1). A nucleoprotein complex is then separated from other cellular constituents (stage 2). This complex is dissociated and the protein is removed (stage 3) and, finally, the nucleic acid is precipitated from the resulting solution (stage 4). Disintegration of... 

In the past, dissociation of the nucleoprotein complex has been brought about by salt solutions or by heat denaturation,129 but, more recently, decomposition has been effected by hydrolysis with trypsin,126 or by the use of dodecyl sodium sulfate130 or strontium nitrate.131 Some virus nucleoproteins are decomposed by ethyl alcohol.132 This effect may be similar to that of alcohol on the ribonucleoproteins of mammalian tissues. If minced liver is denatured with alcohol, and the dried tissue powder is extracted with 10% sodium chloride, the ribonucleoproteins are decomposed to give a soluble sodium ribonucleate while the deoxyribonucleoproteins are unaffected.133 On the other hand, extraction with 10 % sodium chloride is not satisfactory unless the proteins have first been denatured with alcohol. Denaturation also serves to inactivate enzymes of the tissues which might otherwise bring about degradation of the nucleic acid during extraction. 

G. Scholes, The Radiation of Chemistry of Aqueous Solutions of Nucleic Acids and Nucleoproteins, in Progress in Biophysics, Vol. 12, Pergamon Press, London, 1963. 

With chromatin suspensions, DNA product yields are an order of magnitude lower as compared to DNA solutions. This is partially due to OH-scavenging by the nucleoproteins, but since one deals here with suspensions, OH-scavenging by dissolved impurities has an even more dramatic effect than in DNA solutions. Moreover, some of the DNA damage maybe repaired by the surrounding protein, but the existing data do not provide a firm conclusion concerning this point. 

Carbohydrates have long been thought to be associated with the nucleic acid of the bacilli. Bendix ° prepared from the intact cell a nucleoprotein fraction which, after hydrolysis, yielded derivatives which were reducing to Fehling solution and gave a green coloration in the orcinol test. Bendix thus claimed that pentoses were present in the nucleoprotein of M. tuberculosis. 

The solubilization of j8-D-fructofuranosidase bound to cell walls of sugar beet Beta vulgaris) seedlings has been studied.Gel filtration etc. studies confirmed that substances which were released from the cell wall together with bound jS-D-fructofuranosidase acted on the enzyme in dilute salt solutions and caused insolubilization of the enzyme. The characteristics of two different fractions of the substances were analogous to those of nucleic acids and nucleoproteins. 

Nucleoprotein complex can be extracted from cells with N NaCl solution. If the resulting viscous solution is shaken with chloroform containing a little cetyl alcohol, the protein forms a gel at the chloroform/water interface and the sodium salts of the nucleic acids remain in the aqueous phase. Chromatography and centrifuging can then be used to isolate pure specimens (Chapter 14.3). Samples of DNA can be dissolved in water to form very viscous solutions. On adding alcohol to these, a soggy cotton wool-type of precipitate is obtained, from which semi-crystalline threads of DNA can be picked out. 

Nucleoprotein complexes in biological specimens can frequently be separated into their components by fairly simple methods. In the presence of concentrated phenol and a detergent, for example, a cell homogenate will form two liquid phases. Proteins are denatured and become insoluble in the aqueous phase, while the nucleic acids remain soluble. Alternatively, the separation of protein and nucleic acid components from an aqueous NaCl solution can be effected with chloroform (Chapter 11.4). 

Aside from whether they are directly active per se, or merely as precursors of active metabolic intermediates, carcinogenic polycyclic hydrocarbons can initiate carcinomas (e.g. epithelioma) at the point of application on the skin (the commonest mode of study), or can lead to distant tumours, as in bronchogenic cancer [23]. The mechanism probably includes solubilisation of the carcinogens (by proteins, nucleoproteins, fatty acid esters, etc. or even in aqueous solution [58]), so as to enable them to traverse the dermis (possibly via an external solvent) and thus react in the epidermis, which possesses a lipoid barrier. Alternatively, appendageal transport routes (hair follicles, sweat glands, sebaceous glands) may be used [59]. When carcinogens act at a distance, as with liver, breast, bladder and other internal cancers, an internal transport mechanism or medium is evidently needed. 

Low-angle neutron scattering A set of techniques that can be used to find the size of a particle in solution or to find the size or spacing of internal regions that can be distinguished by different neutron scattering power, such as the protein and nucleic acid components of a nucleoprotein particle or labeled proteins within a multisubunit complex. 

When bile is acidified a dense precipitate appears, consisting of nucleoprotein, which is soluble in glacial acetic acid, and mucin, which is insoluble in the acid. The proportions of these proteins differ in different animals. Ox bile is rich in nucleoprotein, but poor in mucin. By careful precipitation the proteins can be precipitated, filtered off, washed, and redissolved in sodium carbonate solution. They give the tests characteristic of their type (p. 130). 

By use of ice-cold 5% trichloroacetic acid and hot ether-alcohol solutions, it was determined that approximately 80% of the activity was found in the acid-soluble and 20% in the nucleoprotein fractions. (Approximately 40% of the activity of the nucleoprotein fraction was present in the nucleic acids.) lodoacetic acid decreased while cyanide increased the rate of uptake of radiophosphorus in the nucleoprotein fraction. Once the yeast cells had incorporated P, it was not lost by resuspending the cells in a radiophosphorus-free medium. Hevesy et al. 

Fig. 1. Schematic representation of isopotential specific volumes, Vj, and partial specific volumes,, of non-conjugated proteins and nucleic acids in an aqueous solution containing inorganic salt (from [89D1]). The difference between isopotential and isomolal specific volumes (Vj > V2 ) i more pronounced with nucleic acids than with proteins. Open circles show V2 C3 = 0, and filled circles indicate Vj at C3 = 0.2 M salt (e.g., NaCl). Since a solvent component concentration of C3 = 0.05 - 0.2 M is usually applied in biochemical experiments, and Vj is the proper volume quantity usually needed in experimental work, v should be used preferably. Because of the slight difference between Vj and V2, in the case of nonconjugated proteins Vj may be used instead of Vj. For nucleic acid studies, however, only use of Vj makes sense. It is obvious that conjugated proteins with pronounced electrolyte character (e.g., nucleoproteins) will show an intermediate behavior, though sufficient data are not available at present. Reprinted from Colloid Polymer Science, Vol. 267, H. Durchschlag, Determination of the partial specific volume of conjugated proteins, pp. 1139-1150, 1989, with permission finm Steinkopff, Darmstadt.

---