Inactivation Of RNase Research Articles

Modification of Bovine Pancreatic Ribonuclease A with 4-Sulfonyloxy-2-nitrofluorobenzene: Isolation and Identification of Modified Proteins

Abstract The inactivation of RNase A that accompanies dinitrophenylation at pH 8 is in major part due to substitution at lysine-41. The amino group of this lysine residue has been postulated to be part of, or close to, the site responsible for binding the phosphate moiety in the substrate. In elaboration of this concept, the consequences that derive from the replacement of 1-fluoro-2,4-dinitrobenzene (FDNB) by a negatively charged reagent with similar chemical and steric properties have been explored. This reagent, 4-sulfonyloxy-2-nitro-fluorobenzene (SNFB), inactivates RNase A at pH 8 by substitution at position 41. It also attacks the enzyme at the α-amino group to produce 1-α-sulfonyloxynitrophenyl (SNP)-RNase A, a fully active derivative. Dinitrophenylation at lysine-41 occurs about 7 times faster than at the α-amino group. By contrast both of these amino groups are about equally reactive toward SNFB at pH 8, an observation interpreted in terms of an oriented approach of the reagent in a configuration unfavorable for reaction at lysine-41. Both 1-α- and 41-SNP-RNase A have been isolated and the locations of the substituents identified by degradative procedures similar to those used in previous studies of the corresponding dinitrophenyl-derivatives. These monosubstitution products and 1-α,41-e-bis-SNP-RNase A, which was also isolated and identified, constitute the principal products formed in the initial stages of the reaction. In contrast to the behavior with FDNB, subsequent reaction at position 7 either does not take place or is strongly suppressed with SNFB. This behavior indicates that 41-dinitrophenyl- and 41-SNP-RNase A may exist in different conformations, with the difference ascribable to the affinity of the sulfonate moiety in the SNP group for the phosphate-binding site. SNFB has a number of advantages over FDNB as a reagent for the modification of RNase A. These include a higher specificity for amino groups, convenient solubility, and the fact that spectra of the reagent and the derivatives that it forms do not overlap as much as with FDNB. The disadvantage of lowered reactivity toward amino groups is offset by the feasibility of studies at relatively high pH values.

Mechanism of inhibition of ribonuclease by bentonite.

A study of the mode of inhibition of RNase by bentonite showed that the inactivation of the enzyme could not be attributed solely to an ion-exchange effect as previously postulated. Vermiculite, which is a clay with higher cation capacity than bentonite, showed very little inactivation of RNase. The spatial arrangement of the clay matrix must also play an important role in accommodating the enzyme molecule. Inactivation of the enzyme by photooxidation did not alter the retention of RNase by bentonite.

Der Einfluß der Temperatur auf die Strahlenempfindlichkeit von Ribonuclease

The radiosensitivity of dry ribonuclease was determined at various temperatures ranging from 90 °K to 300 °K and using 60Co gamma-radiation, 2 MeV protons, and 2 MeV deuterons. The cross section for the inactivation of RNase S (T) is, in this range, given as a function of temperature by S(T) =S0+S1·e-Ea/RT. For inactivation of ribonuclease with Co gamma-rays we found S0=0 and Ea=1000 cal/mole; S1= =0.125 Mrad-1 when irradiation is carried out in vacuo, and S1=0.265 Mrad-1 in oxygen. With protons and deuterons the following values were determined: S0=1.28·10-14 cm2, S1=19.5·10-14 cm2, Ea=1050 cal/mole for 2 MeV protons; S0=2.45·10-14 cm2, S1=31·10-14 cm2, and Ea = 1050 cal/mole for 2 MeV deuterons. Furthermore, by analysis of some recent data from the literature we found that the cross section for inactivation by ionizing radiation of various enzymes, bacteriophages, and bacterial spores in the range from 4 °K to temperatures higher than room temperature can satisfactorily be described by the more general equation S(T) =S0+S1·e-E₁/RT+S2·e-E₂/RT, with E1=1 kcal/mole and E2=4 kcal/mole being constant for all objects and for all circumambient conditions tested. This correlation between inactivation cross section S (T) and temperature T shows three mechanisms of inactivation to occur in biological objects: one (S0) being independent of temperature, while the two others have apparent activation energies of 1 kcal/mole and 4 kcal/mole, respectively.

Inactivation and Induction of Free Radicals in Dried Trypsin

Although the question of the relative importance of ionizations and excitations for the inactivation of molecules of biological importance has been in the center of interest in radiobiology for many years, it is still unanswered (1). Recently, it has been shown that there seem to be correlations between the yields of electron spin resonance (ESR) centers induced in dried enzymes by ionizing radiations and the corresponding inactivation. Thus, in a study of the inactivation of RNase by Co60 y-rays, Hunt and Williams (2) correlated the yield of one class of radicals with the changes in the radiosensitivity of the enzyme irradiated in vacuum and in oxygen, and after postirradiation treatment with oxygen. The yield of induced sulfur-type radicals was correlated with the yield of free SH groups measured by titration. Henriksen2 has shown that the total yields of ESR centers in dried trypsin, exposed at various temperatures to ionizing radiations with different LET, parallel those for enzyme inactivation under the same conditions (3). Similar studies with ultraviolet light have not been reported. However, it has been shown that ultraviolet light and X-rays produce the same type of ESR signals in certain compounds, whereas they give rise to qualitatively different spectra in some other compounds of biological importance. Thus, Koch et al. (4, 5) reported that ultraviolet light and X-rays produce qualitatively equal signals in cysteine, whereas the two radiations give rise to different signals in a number of other sulfur-containing compounds. Koch and Monig (6) found that ESR centers were induced in bovine serum albumin when exposed to light of wavelength 254 m, and, furthermore, that the ESR spectrum of X-ray-induced radicals in this compound changed shape, without a concomitant change in the total number of radicals, on subsequent exposure to ultraviolet light of wavelength longer than 3000 A. The purpose of the present work is to elucidate possible relationships between

Relationship between ribonuclease and certain anionic polymers in utilization of mevalonic acid by liver homogenates.

SummaryLiver homogenates preincubated with RNAse, in contrast to controls, do not incorporate mevalonic acid into non-saponifiable material or cholesterol. This inactivation by RNAse is preventable or reversible with an autoclaved extract of liver or with certain anionic polymers including polyethylene sulfonate, heparin, DNA and even RNA. The polymers also inactivate RNAse in vitro at comparable ratios of inhibitor to enzyme used in the biosynthetic experiments. Inactivation of RNAse in vitro is not observed, however, with comparatively high levels of liver extract. It is concluded that liver extract does not function in the homogenate system merely by inactivation of RNAse but this interpretation may be applicable to the anionic polymers.