Indeed there are radicals, but they are not free.
Perhaps a better terminology for them would be "fixed radicals".
Species such as the disulphide radical have been well established by
spectrophotometric and other evidence (M. Weik et. al. and others). It
is quite remarkable actually how long-lived different radical species
can be a ~100K. The most extensive literature on radical states at low
temperature is the ESR field. Much of it is quite old, but still
accessible in textbooks, such as Box (1977).
Still, I think my best answer to your first question (Bart) is that
cryo-cooled samples are solids, and solid-state chemistry is different
from liquid-state chemistry. This fact is further supported by
observations of the effects of so-called "radical scavengers" under
cryo. Some of these still work in the cold, others do not, and recently
it has been reported (In a talk at RD5) that some scavengers can be
effective against damage to one part of the protein, but not others,
even for chemically identical sites. Indeed, chemically identical sites
in the same protein are often seen to decay at wildly different rates
with or without scavengers. Since two disulphide bonds have exactly the
same likelihood of interacting directly with an x-ray photon (called the
x-ray absorption cross section), these different decay rates cannot be
explained this way. It can also not be explained by "solvent
accessibility" as first pointed out in Burmeister (2000).
So, basically, the model that liquid-phase chemistry is still going on
under cryo is inconsistent with almost every observation made in
cryo-cooled samples. The kinetics in particular make no sense at all
(first pointed out by R. Thorne, and also detailed in Holton, JSR
2007). The only thing solid-state and liquid-state radiation damage
have in common is that some of the chemical consequences (such as
disulphide breakage) are similar.
The "jumping radicals" you describe actually fall very nicely into the
quasiparticle category I was talking about. That is: migration of a
chemical state with no mass transport. Believe it or not, the problem
right now is that there are so many quasiparticle mechanisms that can do
this we still aren't sure which one is involved. The only thing that is
fairly certain is that molecular diffusion is not.
Most of what we know about solid-state chemistry comes from the
semiconductor field, where the reactions are VERY well understood.
Unfortunately, that literature is rather opaque to biologists who aren't
working at a synchrotron. ;) The most well-studied quasiparticles in
silicon are the so-called "electrons" and "holes". The "electrons"
aren't really electrons, but rather a charged excited state of a silicon
atom in the lattice. I will admit that the term "diffusion" is used in
this field. This is largely because these "jumping" quasiparticle
entities moving about a silicon crystal lattice actually obey the ideal
gas law! However, since these are quasiparticles and not real
particles, using the term "diffusion" gives biochemists the wrong
impression of the mechanism.
I know the "primary and secondary" damage formalism, but I think the
problem these days is that I, for one, am fairly convinced that noone is
ever going to be able to observe primary damage. The effects we
classify as "secondary damage" outnumber these reactions by thousands to
one. Colin Nave just described this in his posting, so I won't repeat
it here, but the primary consequence of any ionizing radiation is to put
a whole bunch of atoms into chemically excited states. How this
chemical energy dissipates depends on the structure of the material, and
this makes it hard to predict if you don't know the structure.
-James
Bart Hazes wrote:
Hi James,
We used to talk about primary and secondary radiation damage. The
former operates at room temperature where free radicals were said to
be formed in solution and diffuse around to damage proteins. Under
cryoconditions this no longer happens, leading to greatly improved
crystal life time, but we still have primary radiation damage, with
the photons directly hitting the protein.
It was my understanding that this was still considered to form a free
radical at the affected atom without there being any diffusion
involved. Sulfur atoms would be more sensitive as they have a larger
X-ray cross-section or because they may act as free-radical sinks
where free radicals generated nearby strip an electron from the
sulfur, thereby satisfying their own electronic configuration and
converting the sulfur into a radical state. For instance, in
ribonucleotide reductase a tyrosine free radical is formed
spontaneously (using oxygen and an dinuclear iron site) and "jumps"
over 20 Angstrom from one subunit to another to form a thiyl free
radical in the active site. It then "jumps" back to the tyrosine upon
completion of the catalytic cycle. Although we don't know how it
jumps, certainly not by diffusion, there is general agreement that it
does happen.
People have also observed broken disulfides in cryocrystal structures
with the sulfurs at a distance that is too long for a disulfide but
too short for a normal non-bonded sulfur-sulfur interaction. I seem to
remember that this distance was suggested to indicate the presence of
a thiyl free radical. I'm no chemist of physicist so can't evaluate if
that claim is reasonable but if it is then that would be direct
evidence to support the involvement of a free radical state in
radiation damage.
So I guess my questions/comments are
- what are the great many good reasons to think that free radicals do
NOT play a role in radiation damage under cryo.
- although diffusion does not happen below 130K, radicals do appear to
teleport, at least over short distances.
Bart
James Holton wrote:
I don't mean to single anyone out, but the assignment of "free
radicals" as the species mediating radiation damage at cryo
temperatures is a "pet peeve" of mine. Free radicals have been shown
to mediate damage at room temperature (and there is a VERY large body
of literature on this), but there are a great many good reasons to
think that free radicals do NOT play a role in radiation damage under
cryo.
This "assignment" of free radicals to damage is often made
(flippantly) in the literature, but I feel a strong need to point out
that there is NO EVIDENCE of a free radical diffusion mechanism for
radiation damage below ~130K. To the contrary there is a great deal
of evidence that water, buffers and protein crystals below ~130 K are
in a state of matter known as a "solid", and molecules (such as free
radicals) do not diffuse through solids (except on geological
timescales). If you are worried that the x-ray beam is heating your
crystal to >130 K, then have a look at Snell et. al. JSR 14 109-15
(2007). They showed quite convincingly that this just can't happen
for anything but the most exotic situations.
There is evidence, however, of energy transfer taking place between
different regions of the crystal, but energy transfer does not
require molecular diffusion or any other kind of mass transport. In
fact, solid-state chemistry is generally mediated by cascading
neighbor-to-neighbor reactions that do not involve "diffusion" in the
traditional sense. Electricity is an example of this kind of
chemistry, and these reactions are a LOT faster than diffusion. The
closest analogy to "diffusion" is that the propagating reaction can
be seen as a "species" of sorts that is moving around inside the
sample. Entities like this are formally called quasiparticles. Some
quasiparticles are charged, but others are not. If you don't know
what a quasiparticle is, you can look them up in wikipedia.
Some have tried to rescue the "free radical" statements about
radiation damage by claiming that individual electrons are
"radicals". I guess this must come from the "pressure" of such a
large body of free-radical literature at room temperature. However,
IMHO this is about as useful as declaring that every chemical
reaction is a "free radical" reaction (since they involve the
movement of electrons). I think it best that we try to call the
chemistry what it is and try to stamp out rumors that mechanisms are
known when in reality they are not.
Just my little rant.
-James Holton
MAD Scientist
