(Received for publication, June 20, 1995; and in revised form, September 20, 1995)
From the
Ribonuclease Inhibitor (RI) has been purified from pig testis. It contains 30 half-cystines whose oxidation affects its ability to bind and inhibit ribonuclease (RNase). By N-terminal sequence analyses testis RI showed to be identical to that from porcine liver, for which a characteristic all-or-none type of SH-oxidation by 5,5`-dithiobis(2-nitrobenzoic acid) (DTNB) has been reported (Fominaya, J. M., and Hofsteenge, J.(1992) J. Biol. Chem. 257, 24655-24660). Under comparable reaction conditions, testis RI bound to RNase A did not exhibit this particular type of oxidation; instead, bound RI got intermediate oxidation degrees (up to 14 thiols oxidized per RI moiety) without dissociating from RNase. Moreover, RNase bound to partially oxidized RI was able to express some (15%) of its potential activity (active complex). Only when DTNB treatments accounted for complex dissociation (>14 thiols oxidized per RI moiety) the released RI molecules exhibited the all-or-none oxidation behavior. By both kinetic and circular dichroism analyses, conformational changes have been evidenced for the transition from the inactive to the active form of RI-RNase complex. Relaxation of RI-RNase binding without major alterations in RI structure is proposed as responsible for complex activation. The results are discussed in terms of a model for the reversible regulation of RNase activity mediated by the redox status of RI.
Ribonuclease Inhibitor (RI) ()is an intriguing
protein which is practically ubiquitous in mammalian tissues. Its
three-dimensional structure, as well as that of its 1:1 complex with
RNase A, have recently been reported(1, 2) . Although
the first piece of evidence about the existence of this inhibitor was
gained a long time ago(3) , the biological functionality of RI
remains to be clarified yet (for reviews on early work on RI see (4) and (5) ). Its name suggests that RI is involved in
the control of cytoplasmic RNases, thus having a potential role in
determining levels of gene expression (6) . In fact clear
correlations between cellular metabolic states and levels of RI have
been
reported(7, 8, 9, 10, 11, 12, 13) .
Nevertheless, objections have been raised about a cytoplasmic
functionality for RI(14) . The absence of experimental evidence
about the in vivo existence of cytoplasmic RI-RNase complexes,
together with the known high affinity of RI for enzymes of the
noncytoplasmic RNase superfamily(14) , leave open the question
about the environment where RI plays its role, if intracellular or
extracellular. Whichever the answer to this question it might be, or if
eventually both were true, a second question arises. This is derived
from the tight binding between RI and RNase A, which is the model
ligand mostly used to study the interaction properties of RI (15, 16, 17) . The binding between these two
molecules stands out as one of the tightest reported for
protein-protein interactions (dissociation constant in the femtomolar
range)(18, 19) . This adds more interest to the RI
molecule whose amino acid sequence is characterized by its leucine-rich
repeats(20, 21, 22) , a structural motif
found for other molecules involved in protein-protein
interactions(23) . The extremely high affinity between RI and
certain RNases raises the question of which fate could have the
respective RI-RNase complexes. Aside from taking them as dead
complexes, any other functional implication should require to consider
that binding affinity may be modulated(24) . In this sense, the
numerous half-cystines of the RI molecule (around 30 for the different
RIs studied) (20, 21, 22, 25, 26) may be
involved in its regulation. In fact it is well established that these
residues must be as free thiols for RI to exert its inhibitory
activity; in addition, RNase dissociates from RI when complex
preparations are treated with sulfhydryl reagents, p-hydroxymercuribenzoate being the most employed one for such
a purpose(4, 5, 27) . This close relationship
between the status of the SH groups and RI activity allows to propose
some kind of redox control over the RI-RNase complex(24) . In
this context, a reasonable working hypothesis is to consider the
RI-RNase complex as a heterodimeric enzyme, one of its subunits would
be catalytic, the RNase, and the other one would be regulatory, the RI.
Thiol-disulfide exchange, as a general mechanism of enzyme control (28) , might be involved in the proposed regulation. Inactivation of porcine RI by exchanging of its thiol groups with the Ellman's disulfide (DTNB) has been reported(29) . This investigation, performed on free RI, evidenced an interesting ``all-or-none'' mechanism of inactivation by which, in the presence of amounts of DTNB that did not account for a complete oxidation of the thiol groups present in a RI preparation, the resulting RI molecules did not show intermediate oxidation degrees. Instead, a fraction of RI molecules resulted with all their half-cystines oxidized, whereas the rest maintained all of them as free thiols.
According to our working hypothesis, we were interested in studying the effect of thiol-disulfide exchange on RI bound to RNase rather than on free RI. Thus, we report herein the effects of the Ellman's disulfide on the complex between porcine RI and RNase A, showing that RI, while bound, can reach intermediate oxidation degree, at the same time that the bound RNase can express some of its activity. The obtained results provide new ground to the hypothesis about the redox control of RNase bound to RI.
Bovine pancreatic RNase A (type XII-A), DTNB, and dithiothreitol were purchased from Sigma; cyanogen bromide and SDS were from Serva (Heidelberg, Germany); iodoacetic acid was obtained from Merck (Darmstadt, Germany); and formic acid was from Carlo Erba (Milano, Italy). All other chemicals were at least of reagent grade. The buffers were boiled and degassed by bubbling argon both while cooling down and before use. All of them were 5 mM in dithiothreitol unless otherwise stated.
RI eluted
from the affinity column was pooled and immediately desalted by gel
filtration through Sephadex G-25 M (PD-10 column; Pharmacia LKB,
Uppsala, Sweden) equilibrated and eluted with 20 mM Tris-HCl,
pH 8.0, containing 2 mM EDTA, 150 mM NaCl, and 15%
(v/v) glycerol. The pools so obtained were stored at 4 °C in vials
hermetically stoppered by Mininert valves (Alltech; Deerfield, IL)
provided with septa, through which access with a syringe was possible
without exposing the content to the atmosphere. Before storage, a
positive pressure of argon (0.5 Kg/cm) was bubbled into the
vials through the septa. Periodically the inert atmosphere was renewed
in order to minimize spontaneous oxidation during storage.
Figure 1: CD curves extracted by the CCA method (39) as common features among the set of CD spectra mentioned in the text. Each curve has been assigned to a secondary conformation by comparison of its shape with the known CD spectra for the different secondary structures(38) .
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
Figure SI: Scheme I
Whereas can easily be integrated, integration of requires to previously integrate . In both cases, an integral similar to that numbered as 501 in the integral table of ``CRC Handbook of Chemistry and Physics'' (43) must be solved. The exact integrated equation contains a sum of squared and higher order terms that can be discarded because of their nonsignificant effect on the final results. Thus, the following integrated equations are obtained.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
where A =
[complex](number of SH), B = [DTNB]
, m = A - B; zero subindex refers to concentrations at zero
time.
For each DTNB addition, the k and k
values could be obtained by nonlinear regression
fitting of the experimental data (A
versus time) to the following
equation.
On-line formulae not verified for accuracy
where [TNB](t) and
[TNB
](t) are given by and , respectively, and 13,600 refers to the molar extinction
coefficient of TNB(35) .
On-line formulae not verified for accuracy
where g is the average oxidation degree of the reaction mixture. Obviously, g = 2r/30, since each DTNB molecule reacts with two thiol groups (Fig. SI). Let v be the minimum number of thiol groups that must be oxidized in a complex molecule for dissociation to occur. For a complex preparation subjected to DTNB treatment, the percentage of dissociated complex molecules (the dissociation degree) will be given by the summation of the probabilities of finding molecules with a number of modified groups ranging from v to 30. Thus,
On-line formulae not verified for accuracy
where P(m) can be calculated from by using the particular DTNB/complex ratio (r) accumulated in the reaction mixture.
The activation percentage would also be given by if only dissociation accounted for the release of ribonucleolytic activity. But this is not the case (see ``Results''); the formation of active complex species should additionally be considered. Therefore, in order to predict the activation percentage of a complex preparation subjected to DTNB treatment, should be modified by including the contribution of such active complex molecules to the ribonucleolytic activity. This is taken into account in the following equation,
On-line formulae not verified for accuracy
where u is the minimum number of thiol gropus that must be modified in the complex for it to become active, and f is the fractional activity of the RNase in the active complex relative to its activity as free enzyme.
Figure 2: A, silver-stained (44) SDS-PAGE. Lane a, kit of standard proteins, preincubated in the presence of 1% (v/v) 2-mercaptoethanol, whose respective molecular masses are specified in the ordinate; lane b, purified testis RI preincubated in the absence of 2-mercaptoethanol; lane c, purified testis RI preincubated in the presence of 2-mercaptoethanol. The ``smile'' effect of the band in lane b should be attributed to the action of 2-mercaptoethanol diffusing from the neighboring lanes during electrophoresis. B, reverse-stained (33) SDS-PAGE. Lane a, kit of standard proteins of the specified molecular masses; lane b, testis RI preparation subjected to CNBr cleavage as described under ``Experimental Procedures''; lane c, purified testis RI used as control. In all cases samples were preincubated in the presence of 1% (v/v) 2-mercaptoethanol.
When these studies were under way, investigations on thiol-disulfide exchange properties of free RI from pig liver were reported(29) . Although a high similarity between both porcine RI could be expected, we decided to assess this issue by comparing the N-terminal sequence of testis RI with that of liver RI. As already found for the latter(20) , RI from pig testis was not susceptible to automatic Edman degradation, so suggesting that its N-terminal residue was blocked. The amino acid analysis of testis RI revealed the occurrence of two methionyl residues (data not shown). Therefore, we could use the same strategy employed for the determination of the amino acid sequence of liver RI(20) . Thus, CNBr cleavage of testis RI yielded two major peptides of 36 and 14 kDa, which could be separated by SDS-PAGE (Fig. 2B). Two similar peptides were also seen after CNBr cleavage of liver RI(20) . In addition, this pattern can be taken as an evidence of the occurrence of a blocked methionine as the N-terminal residue; upon CNBr hydrolysis this methionine would migrate, as blocked homoserine, with the front in SDS-PAGE. The two peptides, once electroeluted from the gel were subjected to automatic Edman degradation, rendering the following N-terminal sequences: CB1, Asn-Leu-Asp-Ile-Cys-Glu-Gln-Leu and CB2, Leu-Thr-Gln-Asn-Lys-His-Leu-Leu-Glu-Leu-Gln-Leu. These two sequences are identical to the respective N-terminal sequences of the fragments CB2 and CB3 of liver RI(20) . These results constitute strong evidences about the identity of both porcine inhibitors, although they proceed from different organs. Consequently, the knowledge gained in this work about the redox properties of porcine RI bound to RNase A can be taken as an extension of the thiol-disulfide studies carried out with free porcine RI(29) .
Figure 3:
Evolution of the oxidation degree of a
complex preparation subjected to successive treatments with
substoichiometric amounts of DTNB. , moles of oxidized SH per mol
of complex, as calculated from the accumulated increase in A
of a reaction mixture of known complex
concentration;
, moles of SH that remain reduced per mol of
complex as measured by the Ellman's method (see
``Experimental Procedures'');
, mol of oxidized plus
unoxidized SH per mol of complex, as calculated by adding up the two
preceding determinations. Solid lines correspond to the
results of linear regression analyses of the respective data; the dashed line shows the behavior that would correspond to the
oxidation of just one thiol per molecule of
DTNB.
Leaving aside the effects of complex dissociation, one would predict
that at [DTNB]/[complex] ratios for which
dissociation could be neglected, thiol groups of the complex should
result sorted out in their reaction with DTNB; that is, the higher
reactivity of a cysteinyl residue the quicker its modification and vice
versa. Therefore, a continuous decrease of the fitted k and k
values could be expected.
Alternatively, if the SH reactivity was not affected by the complex
conformation, nonsignificant variations in the rate constants should be
expected all trough the modification. However, the observed behavior in
which SH reactivity fluctuations are detected should be interpreted as
a consequence of conformational changes of the complex induced by
thiol-disulfide exchange.
Figure 4: Gradual modification with DTNB: effects on secondary structure (A) and SH reactivity (B) of RI while bound to RNase. The ordinate scales of A refer to conformational percentages for each of the three pure components (Fig. 1) to which the applied CCA method deconvoluted the set of CD spectra calculated for RI (see ``Experimental Procedures''). Deconvolution analyses were restricted to reaction mixtures in which the accumulated [DTNB]/[complex] ratio did not account for significant dissociation degrees. Conformational analyses of mixtures with higher oxidation degrees were hindered by the important CD contributions of disulfide bridges from the released and therefore fully oxidized RI molecules. In B the results of the kinetic analyses summarized in Table 1are plotted in this restricted range of oxidation degrees.
The three-dimensional structure of RI molecule,
both free and bound to RNase, is basically formed by the repetition of
three structural elements(1, 2) : 16 -helices (12
residues long in average), 17
-strands (3 residues long in
average) forming a curved parallel
-sheet, and 32 loops
(containing between 4 and 9 amino acids) connecting the individual
and
segments; in these loops several types of
-turns
are present. From these figures it can be calculated that
-helices
contain 42% of all RI residues, whereas only 11% are in the parallel
-sheet. Although the percentages of
-helix estimated from the
CD measurements are in good agreement with the x-ray results, the same
cannot be said for
-sheet, which is overestimated probably due to
some contribution from bends (see ``Experimental
Procedures'').
Figure 5:
Gradual modification with DTNB of RI-RNase
A complex: effects on RNase inhibition () and RNase-binding
(
). Experimental data are, respectively, expressed as percentages
of the ribonucleolytic activity or the RNase dissociation found for
preparation aliquots subjected to the same DTNB modification and then
treated with 1 mMpHMB; - - -
- and
represent theoretical
activation and dissociation curves, respectively, as calculated by
assuming a random mechanism of SH modification, with u = 5, v = 15, and f = 0.15
(see text); - and - - - -
correspond to the respective theoretical curves with the same set of
parameters but taking into account a random mechanism of SH
modification only for the RI fraction that remains bound to RNase and
assuming an all-or-none mechanism of oxidation for the RI fraction that
results dissociated after each step of DTNB modification (see
text).
Our first model assumed a random oxidation mechanism, in which all the thiol groups in the complex have the same reactivity. According to this model predicted activation and dissociation curves can be calculated by and . Dotted and dashed lines in Fig. 5show, respectively, these predicted curves when f = 0.15, u = 5, and v = 15. Although they fit well, the experimental data up to a global DTNB/complex ratio of 5.0, a clear discrepancy is observed beyond this value. Thus, the experimental data grow slower than the predicted ones as the global ratio increases. It should be noticed that such a discrepancy becomes patent when the dissociation percentage reaches a significant value (>5%). This fact suggests that random modification may not be a valid model for DTNB oxidation of released RI.
The all-or-none type of reaction reported for the DTNB oxidation of free porcine liver RI (29) suggests that in the reaction mixtures the first RI molecules partially oxidized become more susceptible to subsequent DTNB oxidation. Thus, partially oxidized RI acts as a DTNB monopolizer until all of its thiol groups result modified. This particular behavior of free RI may explain the discrepancies of our model. Free RI molecules with some partial oxidation degree will appear in our reaction mixtures as a result of accumulated DTNB oxidation. They would behave as DTNB monopolizers in a subsequent DTNB addition, so lowering the amount of DTNB available to increase the modification degree of the remaining complex molecules. As a consequence, activation and dissociation of complex preparations will be slowed down once they reach accumulated DTNB/complex ratios, which account for a significant dissociation percentage (e.g. r = 5.0).
It is feasible to calculate theoretical activation and dissociation curves in which this monopolizer effect of the released RI is considered. Thus, for each DTNB/complex ratio the fraction of molecules with v or more of their thiol groups modified (free RI fraction) is calculated by using . To determine the activating and dissociating effect of a subsequent DTNB addition, top priority as DTNB consumer is given to this population. For such a purpose, the amount of DTNB that this population consumes to fully modify its remaining thiol groups is subtracted from the DTNB added, so yielding the DTNB which is really available to increase the oxidation degree of the complex molecules. The activation and dissociation percentages can finally be calculated through and by using the ``effective'' DTNB so determined.
In Fig. 5the predicted activation and dissociation curves so calculated are also plotted. The best results were obtained when f, u, and v took the previous values of 0.15, 5, and 15, respectively. The accuracy of the fit supports the proposed model of random DTNB oxidation for RI-RNase complex, in conjunction with the all-or-none type of reaction for released RI. Additional support would require experimental evidence about the presence in the reaction mixtures of complex species showing variable oxidation degrees, whereas released RI, if present, should have all its thiol groups oxidized.
Figure 6: Ionic exchange chromatography (TSK-DEAE 5PW) of partially oxidized RI-RNase A complex. a and b show the elution profiles of unoxidized RI and RI-RNase A complex, respectively; c shows the elution profile of a complex preparation subjected to an accumulated [DTNB]/[complex] ratio of 7.5. Elution conditions are described under ``Experimental Procedures.'' Fractionation of the eluate followed by measurement of both thiol and protein contents of each fraction allowed to determine the average number of free thiols per molecule along the elution profile. The results of these determinations are also shown in c as bar plots.
The role of RI in the regulation of intracellular RNases remains to be proved. In fact, it has been questioned whether RI-RNase complexes have any implication in the catabolism of RNA, or, on the contrary, if their in vitro detection is only an artifact due to organelle disruption during tissue homogenization(14) . Further investigation of this will be required to accurately assess the cytoplasmic location of RI-RNase complexes. On the other hand, an extracellular role of RI in the regulation of noncytoplasmic RNases has been proposed(14) , which raises questions about how RI is transported out of the cytoplasm.
Any consideration about a potential regulatory role of RI would benefit from knowing the mechanism, if any, through which the inhibitory activity of RI can be modified. As mentioned previously in the introductory statement, RI is a good candidate to be controlled in vivo by thiol-disulfide exchange reactions, in response to changes in cellular redox status. Whether or not such a mechanism of regulation can be viable depends on both the susceptibility of RI to sulfhydryl oxidation and the susceptibility of oxidized RI to reduction (45) . Studies of the first issue, performed on free porcine RI(29) , indicated an extremely high susceptibility of this molecule to become totally oxidized (15 disulfide bridges resulting from its 30 thiol groups) and inactivated through an all-or-none type of reaction. The resulting RI is irreversibly denatured. Hence, in our hands, no reduction treatment was able to restore any of the inhibitory activity lost by reaction of free RI with DTNB, although the employed treatments were effective in the reduction of the 15 disulfide bridges (data not shown). At a first glance, these results seem to be discouraging as to the consideration of some type of reversible in vivo control of RI mediated by the intra- or extracellular thiol-disulfide redox status. But if this control really occurred, one should take into account the effect of thiol-disulfide exchange, not on free RI, but on the RI-RNase complex, which supposedly would be the species candidate for the in vivo control of the ribonucleolytic activity.
The results presented
herein demonstrate that RI bound to RNase is oxidized by DTNB in a
different way from that found for free RI. Thus, partially oxidized RI
can be obtained when preparations of the RI-RNase complex are treated
with substoichiometric amounts of DTNB. This partially oxidized RI
(with up to 14 thiol groups oxidized) remains bound to RNase, only
inhibiting a fraction of its activity (85%). In this complex showing
ribonucleolytic activity (active complex), the RI has an altered
conformation, as deduced from circular dichroism measurements (Fig. 4). The most remarkable fact of the conformational change
associated with the transition from inactive complex to active complex
is an increase in the percentage of -helix at the expense of a
decrease in that of
-sheet and bends. These results are compatible
with the recently reported data about the three-dimensional structure
of free RI (1) and RI-RNase complex(2) . These studies
have shown that the binding of RNase A takes place in an extensive RI
area, which is mainly formed by the parallel
-sheet and loops
connecting
-strands and
-helices. Therefore, the loss of
-sheet/
-turns detected at the activation of the complex could
account for some relaxation of the binding between RI and RNase, which
would explain how RNase can exert some of its potential ribonucleolytic
activity without dissociating.
Our preliminary results on the reversibility of the effects of DTNB oxidation (data not shown) allow us to anticipate that some of the ribonucleolytic activity released from the complex, seemingly that from the active complex, can be reinhibited by reduction treatments. More detailed studies on this particular, currently under way, are required, but for the moment a suggestive hypothesis arises about the possibility of considering the active complex as one of the forms between which the RI-RNase complex can be reversibly switched by the redox status. Fig. 7summarizes a model based on the results obtained that account for this hypothesis.
Figure 7:
Thiol-disulfide exchange of RI-RNase
complex. As a result of oxidizing treatments, pairs of thiol groups of
RI bound to RNase are progressively transformed into intramolecular
disulfide bridges. The RI subunit has 30 thiol groups that can
eventually be oxidized into 15 disulfide bridges; for the sake of
clarity, not all of them have been represented. While the number of
formed disulfides bridges is kept below eight, RI can remain bound to
the RNase subunit. If this number is in the range of three to seven, RI
is still able to mostly inhibit RNase; however, a small fraction (15%)
of the potential RNase activity is expressed. This fractional activity
accounts for the proposed denomination of this partially oxidized
complex (ACTIVE COMPLEX). The release of ribonucleolytic
activity without complex dissociation points to some binding relaxation
that can result from the conformational change detected by CD studies.
Such a conformational change corresponds to a decrease in
-sheet/bend content of RI. Both secondary elements are involved in
the interaction of RI with RNase A(2) ; consequently the
transition from the inactive to the active complex is schematized by a
diminution of the contact areas between both subunits. Binding to RNase
preserves the structure of RI to such an extent that it does not follow
the all-or-none mechanism of oxidation found for free RI. Even more, on
the basis of some preliminary results, we postulate that this structure
preservation could allow reversible transition between the active and
the inactive forms of the complex, depending on the redox conditions.
If oxidizing conditions are further increased, accounting for the
formation of eight or more disulfide bridges, binding relaxation turns
into complex dissociation. Then free RI will exhibit its characteristic
mechanism of oxidation by which it becomes completely oxidized and
irreversibly denatured.
In the scheme shown in Fig. 7,
the formation of disulfide bridges is considered as occurring between
cysteinyl residues which are close to each other in the RI molecule.
This consideration has been derived from the results of the kinetic
analyses carried out for the reaction between RI-RNase complex and DTNB (Table 1). Two values of rate constants were evaluated for each
addition of substoichiometric amounts of DTNB: the second order
constant, k, for the formation of a mixed disulfide between
RI and DTNB and the first order constant, k
, for the
formation of an intramolecular disulfide. Both rate constants fluctuate
only slightly, provided that the complex does not dissociate. Thus, the
second order constant fluctuates around 40 M
min
and the first
order constant around 0.4 min
. One would expect that
DTNB oxidation would yield mixed disulfides only if their subsequent
transformation to intramolecular disulfides was very slow. The
intramolecular reaction will be the rate-determining step if k
< k
[DTNB]. Therefore, mixed
disulfides would accumulate if [DTNB] > 10 mM (0.4 min
/40 M
min
). This is not
the case in the present study, since in order to assess
substoichiometric levels of DTNB, the concentrations of this reagent
were always in the micromolar range. In such conditions, the formation
of intramolecular disulfides is around 3 orders of magnitude faster
than that of mixed disulfides, so explaining that only intramolecular
disulfides are measurably formed. In the study carried out on free RI (29) , the employed DTNB concentrations were also in the
micromolar range, which would explain the disulfide formation also
found.
This formation of intramolecular disulfide bridges allows us to conclude that in the mixed disulfide intermediates the adjacent thiol groups behave as if their ``effective concentrations'' were higher than 10 mM. This should be the consequence of the high number of thiol groups in the RI molecule, which occurs mostly at constant positions in the internal repeats of RI(20, 21, 22) . The susceptibility of RI to form internal disulfide bridges could be considered as an interesting property for its regulation by the cellular redox status. As stated previously(46) , under the reducing intracellular conditions, the oxidation of an intracellular protein should be faster than its reduction if the oxidized form plays some role in vivo. Thus, the formation of intramolecular disulfide bridges in RI can act as a driving mechanism that increases the rate of oxidation in comparison with that of reduction, so allowing both active and inactive forms of the RI-RNase complex to coexist at equilibrium.
The experimental evidence obtained about the existence of an ``active RI-RNase complex'' allows us to maintain that RI, binding RNases, can have a role in reversibly switching them between active and inactive forms. Certainly, the active form may be considered as a poor enzyme, since it only expresses 15% of its potential activity; but, does the cell need the high ribonucleolytic activity that its RNases are able to exert? On the other hand, if the RI-RNase complex really exists in vivo, would the cell ever reach the strong oxidizing conditions required for dissociation? These questions will probably be answered when redox conditions similar to those found in vivo are employed in studying thiol-disulfide exchange of the RI-RNase complex. With this in mind, we are currently investigating the oxidation of the RI-RNase complex by biological disulfides (e.g. GSSG), as well as their thiol counterparts (e.g. GSH) for the reverse reaction.