Nucleic acid succinylation

Succinylated derivatives of nucleic acids may be prepared by reaction of the anhydride with available —OH groups. The reaction forms relatively stable ester derivatives that create car-boxylates on the nucleotide for further conjugation or modification (Figure 1.83). This method has been used in nucleic acid synthesis (Matteucci and Caruthers, 1980) and to derivatize nucleotide analogs such as AZT (Tadayoni et al., 1993). 

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. 

Because derivatization impaired proteolysis, its effect on endogenous ribonclease was studied. Ribonuclease activity was inhibited with increasing succinylation and dropped sharply at an anhydride to protein ratio of 0.8 where 80% of e-NH2 groups of the total protein were succinylated (Fig. 5). Because the ribonuclease was inactivated, an increased nucleic acid content in the precipitated proteins was expected. However, we observed less nucleic acid (NA) in precipitated succinylated protein than in the non-modified controls. Maximum precipitation of succinylated protein occurred around pH 4.5 and contained only 1.8% nucleic acid on a... 

Table VII. The Influence of pH of Protein Precipitation on the Content (%) of Nucleic Acid in Nonsuccinylated and Succinylated Yeast Protein...

Chemical modification of yeast protein has received limited attention though as described above it has potential as a method for facilitating recovery of yeast protein. Current studies are concerned with determination of the functional properties of proteins succinylated during the extraction. The composition of yeast proteins prepared by different methods is shown (Table 8). Noteworthy is the protein and nucleic acid concentration in the yeast isolate which differed from the concentrate in that cell wall material was removed by centrifugation. 

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. 

To achieve success as protein ingredients for food formulation and fabrication, novel proteins should possess a range of functional properties. Frequently during extraction, refining and drying, plant and yeast proteins, intended for food uses, become denatured or altered and subsequently display poor functional properties which render them of limited use. Chemical modification provides a feasible method for improving the functional properties of plant and yeast proteins and potentially may make it possible to tailor proteins with very specific functional properties. In this review the information on modified plant proteins is reviewed and the use of succinylation for the recovery of yeast proteins with low nucleic acid is described. 

Cobalamin enzymes, which are present in most organisms, catalyze three types of reactions (1) intramolecular rearrangements (2) methylations, as in the synthesis of methionine (Section 24.2.7) and (3) reduction of ribonucleotides to deoxyribonucleotides (Section 25.3). In mammals, the conversion of 1-methylmalonyl CoA into succinyl CoA and the formation of methionine by methylation of homocysteine are the only reactions that are known to require coenzyme Bj2. The latter reaction is especially important because methionine is required for the generation of coenzymes that participate in the synthesis of purines and thymine, which are needed for nucleic acid synthesis. 

Pon RT, Yu S. Hydroquinone-0,0 -diacetic acid (Q-linker) as a replacement for succinyl and oxalyl linker arms in solid-phase oligonucleotide synthesis. Nucleic Acids Res 25 3629-3635, 1997. 

Winkler J, Urban E, Losert D, Wacheck V, Pehamberger H, Noe CR (2004) A novel concept for ligand attachment to oligonucleotides via a 2 -succinyl linker. Nucleic Acids Res 32 710-718... 

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