Alkali denaturation

The enzyme is very sensitive to the secondary structure of the substrate. Native calf thymus DNA is quite resistant to enzymic attack by spleen exonuclease, being split at less than 4% the rate at which alkali-denatured DNA is split (11). Long deoxyribonucleotides (average chain length 20-50), which still have complementary double-stranded structure, are rather resistant to the enzyme (26). Some limited results obtained with synthetic polyribonucleotides (11) are rather puzzling since poly C was found to be completely resistant, whereas poly A, poly I, and poly U were degraded at comparable rates. In the solvent used (0.15 M acetate buffer-0.01 M EDTA, pH 5.0), poly A and poly C are believed to have... 

Cyanuric fluoride has been used to modify tyrosine residues, substituting the phenolic hydroxyl group. A maximum of 3 residues in RNase was found to react at pH 10.9 and 25° (148a). However, some mystery surrounds this number, as with other estimates of accessibility, since alkaline-denatured material where all tyrosine residues are available still showed the reaction of only 3 residues with cyanuric fluoride. However, similar observations have been made on iodination in 8 Af urea (11 )- At pH 9.3, Takenaka et al. (149) found that only 2 residues reacted and that 115 was not one of them. Two more reacted after alkali denaturation. Two were resistant under all conditions tested. No enzymic activity data were reported. 

A titration study of a peroxidase from Japanese radish has been reported by Morita and Kameda (1958). The titration curves of native protein, acid-denatured protein, and alkali-denatured protein are dramatically different. Unfortunately only continuous titration curves were obtained, so that an interpretation of the data is not possible at this time. 

For many years, alkali denaturation has been the most popular method for the quantitation of Hb-F in human red cell hemolysates. However, these denaturation techniques have always been subject to question because of interfering properties in the procedure. Chromatographic determination of Hb-F, while providing a separation of the fetal component from the major Hb types, is still subject to error because minor adult hemoglobins may elute together with Hb-F. 

MlO. Matioli, G., and Thorell, B., Kinetics of the alkali denaturation of hemoglobin in the single erythrocyte. Blood 21, 1-7 (1963). 

Singer, H., Chemoff, A. I., and Singer, L., Studies on abnormal hemoglobin. I. Their demonstration in sickle cell anemia and other hematologic disorders by means of alkali denaturation. Blood 6, 413-428 (1951). 

The 5 minutes alkali denaturation step was necessary to permit optimal access of the antibodies to the adduct epitopes on DNA. This step may not be required for detection of other complexes or adducts. 

Combine linearized DNA (50-200 ng) with hexanucleotides (10-fold excess with respect to mass) in 15 pi, boil for 4 min and place on ice/NaCl (except when DNA is added in a molten gel slice in that case it is rapidly cooled to 37°C). A somewhat higher specific activity is obtained if the DNA is alkali-denatured (0.2 N NaOH-I-0.2 M EDTA (final concentration) for 5 min), neutralized by adding 0.1 vol. of 2 M ammonium acetate and ethanol-precipitated. 

Denature DNA as in Al. Alkali-denatured DNA can be applied directly to a Zeta-Probe membrane (but 0.2 vol. of 5 M NaCl should be added to the sample prior to applying to Nytran), but can then not be reprobed (otherwise neutralize before blotting). 

ORD measurements on alkali-denatured cytochrome oxidase did not show Soret Cotton effects in the oxidized state, but a complex spectrum appeared at pH 11.6 in the reduced state, indicating heme-heme interactions. Polylysine-heme complexes were used as models (252). The optical activity of both oxidized and reduced heme a was measured in various solvents and under different conditions (253, 254). 

Examination of a small number of blood samples from patients with various diseases involving blood abnormalities have not shown any of these conditions to be associated with a fetal-type hemoglobin absorption spectrum with the exception of Cooley s anemia, a congenital disease which is common in certain Mediterranean countries. Liquori (1951) has found that blood from cases of this type of anemia has a slow alkali denaturation rate, comparable with that observed for fetal-type hemoglobin by Jonxis (1949), which enables it to be clearly distinguished from the normal adult type, which is denatured much more rapidly under the same conditions. 

The difference absorption spectra of hen and turkey lysozymes in the alkaline pH region had three maxima at around 245, 292, and 300 nm and had no isosbestic points.The ratio of the extinction difference at 245 nm to that at 295 nm changed with pH. These spectral features are quite different from those observed when only L-tyrosyl residues are ionized, and it was impossible to determine precisely the pK values of the L-tyrosyl residues in lysozyme by spectrophotometric titration. A time-dependent spectral change was observed above about pH 12. This is not due to exposure of a buried L-tyrosyl residue on alkali denaturation. The disulphide bonds and the peptide bonds in the lysozyme molecule were cleaved by alkali above about pH 11. The intrinsic pK value of L-tyrosine-23 of hen lysozyme was determined to be 10.24 (apparent pK 9.8) at 0.1 ionic strength and 25 C from the c.d. titration data. Comparison of the c.d. 

The isolated clone is sequenced completely. Suitable restriction fragments are subcloned into pUClS plasmid and sequenced using the universal Ml3 forward and reverse primers or specific synthetic oligonucleotides. DNA sequencing is carried out by the dideoxy chain termination method with [ SJdATP, using Sequenase version 2.0 sequencing kit (Amersham) on alkali-denatured double stranded templates. 

Hemoglobin E shows a striking change in electrophoretic mobihty with change in pH. At pH 6.5 its mobility is greater than that of A and slightly less than that of S, and at pH 8.8 it is nearly identical with that of C. The absorption spectrum, solubility, and lability to alkali denaturation of E are similar to those of A. i... 

Fig. 2. Dot hybridization of cloned sequences from the "140 bp DNA". The single stranded DNA of 26 clones ( 50 ng) woe spotted as indicated in panel D. Controls, alkali denatured prior to spotting were the following a) M13mp8 vector DNA (-50 ng) b) calf thymus total DNA (-100 ng) c) 140 bp DNA" (-50 ng) d) "70 bp DNA" (-20 ng). The probes were "140 bp DNA" (panel A), "70 bp DNA" (panel B) and calf th)mius total DNA (panel C). The 12 strong positive clones are underlined in panel D.

Dahl, S.R Villota, R. Twin-screw extrusion texturization of acid and alkali denatured soy proteins. J. Food Sci. 1991,56, 1002-1007. 

It was also observed (4, 5) that the acetylation of the phenol group, and the inactivation, became slower with a lowering of pH of the medium and virtually stopped below pH 4.0. Due to the peculiar nature of pepsin, the pH could not be raised above 6.3 without encountering alkali denaturation. 

For native t-RNA in aqueous solution it is commonly assumed that about half of the bases are exposed to solvent or are not engaged in intramolecular hydrogen bonding, while for alkali-denaturated DNA the percentage of bases in a similar state is certaintly greater. 

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