Investigation of radical‐based damage of RNase A in aqueous solution and lipid vesicles

Abstract

The gamma-irradiation of bovine pancreatic ribonuclease A (RNase A) in aqueous solution were investigated at different doses by vibrational spectroscopy as well as enzymatic assay, electrophoresis, and HPLC analysis. Both functional and structural changes of the protein were caused by attack of H(*) atoms and (*)OH radicals. In particular, Raman spectroscopy was shown to be a useful tool in identifying conformational changes of the protein structure and amino acidic residues that are preferential sites of the radical attack (i.e., tyrosine and methionine). After partial structural changes by the initial radical attack, the internal sulfur-containing amino acid residues were rendered susceptible to transformation. By using the biomimetic model of dioleoyl phosphatidyl choline vesicle suspensions containing RNase A, the damage to methione residues could be connected to a parallel alteration of membrane unsaturated lipids. In fact, thiyl radical species formed from protein degradation can diffuse into the lipid bilayer and cause isomerization of the naturally occurring cis double bonds. As a consequence, trans unsaturated fatty acids are formed in vesicles and can be considered to be markers of this protein damage.

Structural lesion to ribonuclease A caused by reductive stress: Assessment by Raman spectroscopy

“…This conclusion was drawn out from the analysis of the doublet at 850-830 cm −1 that depends on the state of the hydrogen bonding involving the OH group of Tyr [4]. This intensity ratio, average of all six Tyr residues, was about 0.8 in untreated RNAse A t-BuOH-containing aqueous solution, indicating the presence of three or four "buried" Tyr ( Fig.…”

“…1(a)). The I 850 /I 830 ratio increased of ≈30% after 23 Gy irradiation, indicating that one or two Tyr residues are located in a more [4].…”

“…In fact, the H • -induced desulfurisation involving these residues yield α-aminobutyrric acid (Abu) and Ala (reactions (5) and (6)). In addition, the reductive radical attack towards Met residues has been recently found to yield diffusible sulfur radicals able inducing damages in cellular membranes [4][5][6]. ,…”

“…In a previous paper both the functional and structural changes of bovine pancreatic ribonuclease A (RNAse A) have been analysed under mainly oxidative radical conditions ( • OH 90% and H • 10%) and a stronger reactivity involving in particular sulfur-containing residues and aromatic residues has been shown [4]. Since recently the reductive stress has been placed in the context of a biological damage, constituting the basis of tandem-lipid damage the separate contribution of a reducing species such as H • to the changes produced in RNAse was investigated more in detail in the present work [5,6].…”

Spectroscopic and chemometric approaches to radiobiological analyses

“…Early applications of both techniques in radiobiology had focussed on studies with Raman spectroscopy of radiation-induced peroxidation in model phospholipid and membrane systems [46,47], structural changes in protein [48] and in nucleic acids [49]. Such studies continue to contribute to the understanding of the interaction mechanisms between radiation and key molecular species and structures within the cell [48][49][50][51]. These investigations have since evolved to Raman spectroscopic studies of the effects of in-vitro proton irradiation of excised tissues [52], and radiotherapeutically irradiated (γ-radiation) tissue specimens [53][54][55].…”