Ricin & Abrin

Often featured in the media as absolutely lethal and capable of mass destruction, the plant toxin ricin, and its congener abrin are some of the most poisonous substances known to science. Today we are going to explore their deadly secret, which is the chemistry behind their mechanism of action. 

 

Ricinus communis

Abrus precatorius

Ricin and abrin are unlike what we have previously encountered, which are mostly alkaloids. Both ricin and abrin are protein toxins, and they are huge molecules. Weighing in at approximately 64 – 65 kilodalton (g/mol), they are 225 times larger than belladonna's toxin atropine. As proteins, ricin and abrin are made up of chains of amino acids, which are folded into a 3-dimensional structure  the shape of a globule (ball-like). Ricin and abrin are mostly similar, and differ only in the arrangement of certain amino acids. They have identical mechanism of action, so you can treat both as equivalent. It should also be noted that slight variations in amino acid sequences give rise to different isotypes of ricin and abrin depending on different plants, but that's not a concern. Ricin and abrin are classified as toxalbumin (albumin is a protein) or lectin (plant-based protein that can bind to sugar). Ricin consists of two protein chains called Ricin Toxin A (RTA) and Ricin Toxin B (RTB), which are linked together by a disulfide bridge (Figure 1). Neither RTA nor RTB are toxic on their own, but the two chains facilitate each other to become deadly. Hence, we refer to this type of toxins like ricin and abrin as A-B toxins, A stands for ‘active’; B stands for ‘binding’. In fact, A-B toxins are more common in bacteria than in plants, as they include some of the most lethal (by LD50) substances known to humans like botulinum toxin (Botox), tetanus toxin, anthrax toxin etc. 

Figure 1: 3D Protein structure of ricin. 3 domains of RTA and 2 domains of RTB are coloured differently. Polito, L.; Bortolotti, M.; Battelli, M. G.; Calafato, G.; Bolognesi, A. Toxins. 2019, 11, 1–16.

Hindered by their large sizes, ricin and abrin cannot diffuse across animal cells effectively like simpler plant toxins. In order to enter a cell, RTB binds to sugar molecules called glycoproteins that are located on a cell's surface. RTB contains two active sites called domains that specifically bind to the sugar lactose (when it was first discovered), hence a lectin. The effect is like ringing the doorbell of a cell for an invitation. The cell then grants ricin (abrin) entry via a process called endocytosis, in which the ricin molecule gets 'eaten' by the cell. Once inside, the cell's machinery (called Golgi apparatus) cuts RTA loose from RTB by digesting the disulfide bond. There goes mayhem because RTA is the knife in the lollypop! Once again, we have seen how Mother Nature fashions toxins in creative ways such that the victim's own body activates the toxins to their doom.

Since today’s post is all about chemistry, here's how ricin (abrin) works. RTA is itself a globular protein with catalytic activity called N-glycosidase. Whenever we deal with a catalyst like ricin, it means something that facilitates or promotes a chemical reaction but is itself not consumed in the process. In other words, ricin and abrin are different from other plant toxins because they either bind to a target or react with them to form a permanent chemical bond. This makes them all but more deadly because the toxins are always regenerated and  free to do damage again and again. Examine Figure 2 carefully and as always, follow the coloured arrows.

 

Figure 2: Ricin toxicity, simplified mechanism.

Ricin and abrin stop the body's ability to make proteins by inactivating ribosomes, which are made up of RNA. Zoom into an individual RNA molecule and you see three components, i.e., a phosphate backbone that links the RNA chains, a ribose sugar, and a nitrogenous purine base (either A, T, G or U, find out what they are if you're not sure). If you can break any of these, you break a ribosome. This is what RTA does, it breaks the covalent bond between the ribose sugar and the purine base adenine (A), the part I coloured pink. Remember I mentioned N-glycosidase? N- means the nitrogen of adenine, glyco- means sugar; -ase means catalyst. The reaction itself is called depurination and RTA has three domains that constitute an active site where catalysis occurs. The active site contains amino acids (bolded in black) that will split apart a water molecule (your body is 70% water) and use it to break the RNA's ribose-adenine bond. I’ve simplified the active site by showing only amino acid side chains in a half circle, you can think of the actual case as RTA wrapping around an RNA (see Figure 3). An amino acid snatches a hydrogen atom from water, making a transient electron rich hydroxide ion that attacks the carbon atom-1 in the ribose molecule. At the same time, another amino acid donates a hydrogen atom to the adenine's nitrogen, which is bonded to the aforementioned carbon-1. Hence, a new bond is made for both the ribose and the adenine (coloured in blue and pink, respectively), while the old sugar-adenine bond is broken. Bye-bye ribosome! 

 

Figure 3: Ricin toxicity, cellular pathway. Franke, H.; Scholl, R.; Aigner, A. Naunyn. Schmiedebergs. Arch. Pharmacol. 2019, 392, 1181–1208.

 

What’s more, compare the structure of RTA before and after the depurination reaction, it's essentially the same! RTA is a catalyst and it can inactivate thousands of molecules of ribosomes in a matter of minutes. Ricin and abrin kill by causing multi-organ failure because every cell, tissue and organ rely on ribosomes. It takes only 1.5 milligrams of ricin to kill an adult human if injected or inhaled, but ingestion appears much less toxic perhaps because ricin is poorly absorbed by the human body. Our gut contains acid and enzymes that may break down some but not all ricin. 1.5 milligrams of ricin may not sound like much, but it actually contains 1.3 x 10^16 molecules, and one molecule of ricin is enough to kill a cell (our body contains about 30 trillion cells). On the other hand, abrin is even more toxic, about 70 times more lethal than ricin based on animal experiments. This could be due to extra catalytic potential or stability of abrin in the body. Although no specific antidote is available, both abrin and ricin will eventually degrade and get excreted overtime. Cells contain enzymes like lysozyme that can breakdown the peptide bonds of toxalbumins. Victims can and do survive given extensive symptomatic treatments. If ricin and abrin are given in a sublethal dose, the body can produce antibodies against the toxalbumins, much like how we develop acquired immunity towards bacterial toxins or snake venom. Ricin and abrin are currently classified as potential bioweapons of mass destruction by the Biological and Toxins Weapon Convention (BWC). Considering their threat to humanity when misused, military research are developing vaccines for both toxins. Personally, I don't know if that's necessary at all!

 

Finally, I have a confession before I end.  The chemical mechanism in Figure 2 is over-simplified and it’s not accurate. Below is what’s really happening, and that RTA stabilises the depurination reaction by favouring the formation of an oxycarbenium ion, which is then attacked by water (enhanced nucleophilicity by glutamate side chain). Take this as your homework and see if you can make sense of the actual mechanism of RTA.

 

Figure 4: Ricin toxicity, precise mechanism of action.