Protein with reduced nucleic acid

Isolation of Proteins with a Reduced Nucleic Acid Level. The procedure is virtually identical to that described for succinylation of yeast proteins (87). In a typical experiment proteins, together with NA, were extracted from the disrupted yeast cells at pH 8.5-9.0 and centrifuged at 15,000 rpm for 30 min at 5°C. Citraconic anhydride then was added in small increments to the supernatant with constant stirring while the pH was maintained between 8.0-8.5 by adding 3.5IV NaOH. After the stabilization of the pH, the pH of the solution was decreased to 4.2 to precipitate the proteins. Protein then was separated by centrifugation, dissolved in water (pH adjusted 8.5), dialyzed extensively against water (pH 8.5) at 5°C, and lyophilized. 

The absorption spectra of three yeast protein preparations prepared by different procedures were compared (Fig. 8). The presence of nucleic acid which has a X maximum at 260 nm tend to shift the absorption spectrum of yeast protein to lower wavelengths. The ratio of absorption at 280 to 260 nm is indicative of NA contamination in protein samples a ratio of more than one indicates pure protein devoid of nucleic acid whereas a ratio of 0.65 indicates approximately 30% contamination with NA. The yeast protein extracted with alkali and directly acid precipitated showed a X max at 260, a 280/260 ratio of 0.67 and contained 28%, NA determined chemically. Protein extracted in alkali, adjusted to pH 6 and incubated at 55°C for 3-5 hours, to reduce NA with endogenous ribonuclease, had a X max at 260, a 280/260 ratio of 0.8 and a NA content of 3.3% while yeast protein prepared by the succinylation procedure and precipitated at pH 4.5 showed a X max at 275 nm, a 280/260 ratio of 1.0 and nucleic acid content of 1.8. 

The high content of nucleic acid in yeast is a potential problem associated with the consumption of large amounts of yeast nucleopro-tein in foods. Phosphorylation is one of the methods used for reducing nucleic acid of yeast proteins (Table 2) [67]. Huang and Kinsella [68] reported the effective removal of nucleic acid from yeast proteins by POCl3-phosphorylation. The phosphorylated protein showed improvement in emulsifying activity and emulsion stability, and produced stable but weak foams at neutral pH. The authors also noted that the in vitro digestibility of yeast protein was not affected by phosphorylation as reported for casein [60] and soy protein [65]. 

Fish sperm contain nucleoprotamines. Upon treatment with sulfuric acid, the nucleoprotamines are reduced to nucleic acid and protamine sulfate. The chemically heterogeneous protamines of molar mass 2000-8000 thus obtained contain only a few different kinds of amino acid residues per molecule. They are relatively rich in basic amino acids, as the composition of the protamines clupeine and salmine shows (Table 29-6), and never contain cystine, aspartic acid, or tryptophan. The basic amino acids are responsible for the bonding of the protein to the nucleic acid component. 

Schiff base compounds formed by the interaction of oxidation products with proteins, phospholipids and nucleic acids produce chromophores showing characteristic fluorescence spectra. The Schiff base formed between malonaldehyde and amino acids is attributed to the conjugated structure -NH=CH-CH=CH-NH-. Lipid-soluble fluorescence chromophores are produced from oxidized phospholipids and from oxidized fatty acid esters in the presence of phospholipids. These chromophores have fluorescence emission maxima at 435-440 nm and excitation maxima at 365 nm. The Schiff base of malonaldehyde and phospholipids has a higher wavelength maximum for emission (475 nm) and excitation (400 nm). The interaction between oxidized arachidonic acid and dipalmityl phosphatidylethanolamine produce similar fluorescence spectra (maximum excitation at 360-90 nm and maximum emission at 430-460 nm). The products from oxidized arachidonic acid and DNA have characteristic fluorescence spectra, with excitation maximum at 315 nm and emission maximum at 325 nm. Similar fluorescence spectra, with excitation maximum at 320 mn and emission maximum at420 nm, are obtained from the interactions of either lipid hydroperoxides or secondary oxidation products with DNA in the presence of metals and reducing agents, or different aldehydes, ketones and dimeric compounds from oxidized linolenate. Therefore, the Schiff base produced from various oxidized lipids and phospholipids and DNA may be considered to be due to a mixture of closely related chromophores. 

A selective method of preventing the expression of adhesion molecules or cytokines is the use of antisense oligonucleotides. These oligonucleotides are short sequences of nucleic acids complementary to mRNA sequences of specific proteins of interest. If delivered to the cytoplasmic compartment of cells these oligonucleotides are able to form a complex with their target mRNA. In this way the translation of mRNA into protein by ribosomes is inhibited. The subsequent mRNA degradation by RNAse H results in reduced expression of the protein (see also Chapter 5 for a description of antisense ohgonucleotides as therapeutic modalities). 

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