Nucleic acids separation from proteins

Many of the same models and techniques have been used to study the transitions in these two types of biopolymers, and we will present some common background information first. Then we will specialize and present the results of important thermodynamic studies in proteins and nucleic acids separately. However, common to both reports is the observation that the application of thermodynamic measurements and a thermodynamic analysis to carefully but widely chosen systems allows one to gain insights into structural details that complement molecular structure determinations obtained from instrumental techniques such as spectroscopy and X-ray crystallography. 

The nucleic acid of the tubercle bacillus has been identified largely as deoxyribonucleic acid. This acid has been found to occur in association with polysaccharide fractions, probably due to the remarkable adsorptive powers of the latter. The nucleic acid, which is antigenically inactive (see p. 325), has been successfully separated from protein and carbohydrate material by electrophoretic means. ... 

A separation of the nucleic acid, polysaccharide and protein fractions of tuberculin was effected by the Tiselius electrophoretic technique, which was developed for large scale work in this field by Seibert and Watson. The polysaccharide was relatively immobile and thus was easily removed from the protein and nucleic acid. At pH 5.0 (or less), the nucleic acid and protein traveled in the electrophoretic tube as a single component. At higher pH values, the two tended to move independently. It was probable that some of the protein and nucleic acid was present as nucleoprotein. 

Because the Pol II core alone is sufficient to maintain the transcription bubble and the DNA-RNA hybrid during RNA chain elongation, there must be exposed elements on the enzyme surface that keep the nucleic acid strands apart. Protein elements are needed to separate the DNA strands downstream of the active site and to separate the RNA from the DNA template strand at the upstream end of the hybrid. On the basis of their location with respect to nucleic acids, several Pol II structural elements are predicted to maintain the bubble and the hybrid. These proposals are currently tested by site-directed mutagenesis. Separation of the DNA strands at the downstream edge of the bubble may be attributed to binding of the DNA template strand by switch regions 1 and 2 and to blocking of the path of the nontemplate strand by fork loop 2. In the Pol II-TFIIS complex structure, fork loop 2 is ordered and restricts the cleft to a diameter of 15 A, consistent with the proposal that this loop removes the DNA nontemplate strand from the template strand before the active site. 

Recently, a total serum protein N-glycosylation profiling was attempted on a CE chip by Ehrlich and coworkers [213]. The authors employed a glass chip with a double-tee injector and a 4% linear polyacrylamide sieving medium (analogous to nucleic acid separations). Profiling of serum samples from chronic hepatitis patients identified the differences in N-glycan composition in cirrhotic and noncirrhotic cases, and demonstrated the potential of microchip approach for these types of clinical studies. 

Hydrolysis of nucleoproteins separates the acids from the proteins. Further hydrolysis yields the components of nucleic acids, namely sugars, bases, and phosphoric acid. The nucleic acids differ from each other, depending upon the source, in chain lengths, sequences, and distributions of bases. As in the proteins, the primary structure of nucleic acids is determined by partial and sequential hydrolysis. 

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). 

Acid-soluble sugars, phosphates, nucleotides, and phospholipids are usually removed by taking advantage of the acid and alcohol insolubility of nucleic acids and nucleoproteins. Protein removal or elimination may be accomplished in three ways (1) acid hydrolyms of the nucleic acids to acid-soluble components without protein hydrolysis (S) hydrolysis of protein phosphorus to inorganic phosphorus and of RNA to acid-soluble mononucleotides by alki and (S) dissociation of nucleic acid from protein by salt extraction followed by protein denaturation. The separation of the two types of nucleic acids from each other, which is required in any case depending on ultraviolet spectrophotometry or phosphate analysis, is... 

Each tobacco mosaic virus particle consists of one long thread of nucleic acid embedded in protein. The protein surrounds the nucleic acid in loops or in the fashion of screw threads making up the super-molecule. Treatment with phenol separates nucleic acids from protein. The nucleic acid obtained in this mild way remains infectious, and in a host cell can cause virus multiplication and consequent symptoms of disease. About 95% of the material is protein it consists of individual subunits with a molecular weight of 17,500, which exhibit a marked tendency to aggregate At neutral or slightly acidic pH the protein molecules aggregiite to little rozls, very similar to the intact virus particles both in shape and size. The amino acid sequence is now known. Mutants obtained by nitrous acid treatment (see above) show up differences in the amino acid sequence usually only one amino acid has been replaced, for example, serine by leucine, or leucine by phenylalanine. 

Chemists and biochemists And it convenient to divide the principal organic substances present m cells into four mam groups carbohydrates proteins nucleic acids and lipids Structural differences separate carbo hydrates from proteins and both of these are structurally distinct from nucleic acids Lipids on the other hand are characterized by a physical property their solubility m nonpolar solvents rather than by their structure In this chapter we have examined lipid molecules that share a common biosynthetic origin m that all their carbons are derived from acetic acid (acetate) The form m which acetate occurs m many of these processes is a thioester called acetyl coenzyme A... 

Biopolymers are the naturally occurring macromolecular materials that are the components of all living systems. There are three principal categories of biopolymers, each of which is the topic of a separate article in the Eniyclopedia proteins (qv) nucleic acids (qv) and polysaccharides (see Carbohydrates Microbial polysaccharides). Biopolymers are formed through condensation of monomeric units ie, the corresponding monomers are amino acids (qv), nucleotides, and monosaccharides, for proteins, nucleic acids, and polysaccharides, respectively. The term biopolymers is also used to describe synthetic polymers prepared from the same or similar monomer units as are the natural molecules. 

Lipoteichoic acids (from gram-positive bacteria) [56411-57-5J. Extracted by hot phenol/water from disrupted cells. Nucleic acids that were also extracted were removed by treatment with nucleases. Nucleic resistant acids, proteins, polysaccharides and teichoic acids were separated from lipoteichoic acids by anion-exchange chromatography on DEAE-Sephacel or by hydrophobic interaction on octyl-Sepharose [Fischer et al. Ear J Biochem 133 523 1983]. 

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