{"id":1709,"date":"2010-11-06T07:55:29","date_gmt":"2010-11-06T07:55:29","guid":{"rendered":"http:\/\/thetravellerslastjourney.com\/shai\/2018\/11\/27\/892-revision-v1\/"},"modified":"2019-09-26T09:44:54","modified_gmt":"2019-09-26T09:44:54","slug":"biotinylation","status":"publish","type":"post","link":"https:\/\/thetravellerslastjourney.com\/shai\/2010\/11\/06\/biotinylation\/","title":{"rendered":"Biotinylation"},"content":{"rendered":"

Editorial notes<\/strong>:
\nIn November 2010, Shai begins the second phase of his intellectual journey with an\u00a0essay\u00a0dedicated to scientific summation. Throughout 2010 Shai\u2019s writings are focused primarily on book reviews with the occasional venture into reviews of science. This focus shifts in 2013 when Shai produces 24 science-related compilations. Between November 2010 and June 2016, Shai produces\u00a060 write-ups<\/a> related to varied scientific matters.<\/em><\/span><\/p>\n

Biotinylation is the addition of\u00a0biotin<\/a>\u00a0to a molecule. It is often used by biologists studying how proteins move around in cells, or to grab some molecules in the cell to study.<\/p>\n

Who is this for?<\/b><\/p>\n

Everyone<\/a>. But given that this is a fairly\u00a0specialized<\/a>\u00a0topic I have written this in sections of increasing technicality. If you find yourself zoning out, please skip to the last paragraph before\u00a0clicking away<\/a>. Even if you’re not a\u00a0molecular biologist<\/a>, and even if you don’t have any science background, please give the next few paragraphs a go, they were written for you. \ud83d\ude42<\/p>\n

A simple introduction<\/h3>\n

All men by nature desire knowledge.\u00a0Aristotle<\/a><\/p><\/blockquote>\n

In science we don’t see what we study.\u00a0Pathologists<\/a>\u00a0study diseases, but they never see them, all they see are sick patients or animals, and even then, they don’t actually see their “sickness”, what they see instead are markers of illness. For example, a scientist studying\u00a0Alzheimer’s Disease<\/a>\u00a0could see an old man displaying\u00a0dementia<\/a>\u00a0or a dissected brain full of\u00a0plaques<\/a>\u00a0(bits of the brain that have aggregated). This principle extends to almost everything we can study. A sociologist never sees poverty itself. A chemist never sees a molecule. A physicist never sees gravity. I think you get the point.<\/p>\n

You can probably begin to tell that there are reasons for this gap between what we study and what we actually observe. One reason is that the thing we study is a word given to a collection of things or a state of affairs. For example, something like “drug addiction” could be broken down into simpler ideas like dependency, compulsiveness, and so forth. Another reason is that some of the things we study depend on a first-person perspective. So if I were to study the psychology of kindness I’d have to use some sort of proxy measure, like charity given per year. And finally, often what we study is something too small (e.g. cells in the body) or far (e.g. planets outside the solar system) to see directly and so we use indirect measures instead.<\/p>\n

Biotinylation is a tool used (especially but not exclusively) by biologists who study how\u00a0cells<\/a>\u00a0behave. It is a tool that lets us paint – using\u00a0biotin<\/a>\u00a0– something we’re interested in, and then later we can collect that thing by selecting all the biotin. Let’s have an analogy of sorts: let’s say I have a cage full of African swallows and a cage full of European swallows, and I want to put them in the same cage to see if they will fight. Because they all look the same I need to tag one population with biotin paint, and then later I can separate the two groups based on which are and are not painted.<\/p>\n

So far so good, but what if the swallows are too small to see which are painted with biotin? Then I’d need a sticky blanket which only sticks to biotin. So if I wrap all the swallows in this sticky blanket, the ones that can fly away are obviously the ones that were never painted, while all those ones who stay stuck to the blanket are stuck because they were painted with biotin. Would you believe it, is such a thing, and it\u2019s called\u00a0streptavidin<\/a>. Streptavidin is a molecule which grabs on very strong to biotin, so strongly in fact that whatever the biotin is attached to will also be stuck to the streptavidin.<\/p>\n

Why why why?<\/h3>\n

Nothing tends so much to the advancement of knowledge as the application of a new instrument. The native intellectual powers of men in different times are not so much the causes of the different success of their labours, as the peculiar nature of the means and artificial resources in their possession.\u00a0Sir Humphrey Davy<\/a><\/p><\/blockquote>\n

The story goes that in 1927\u00a0Margaret Boas<\/a>\u00a0was conducting an experiment that involved feeding rats raw eggs. Margaret observed that the rats developed some sort of\u00a0malnutrition<\/a>\u00a0and that they could be rescued by feeding them\u00a0vitamin H<\/a>, which was later shown to be the same thing as biotin. Although Boas didn\u2019t know it at the time, the malnutrition suffered by the rats was due to\u00a0avidin<\/a>\u00a0in the egg whites binding all available biotin leaving little available for the animals’ nutritional needs. It took until 1942 for\u00a0Edmond Snell<\/a>\u00a0to provide a more complete identification of avidin and its ability to bind biotin strongly. (The name avidin comes from its\u00a0avid<\/u>\u00a0binding to biotin<\/u>). The binding capacity of avidin for biotin is very strong (Kd<\/sub><\/a>\u00a0~10-14<\/sup>-10-16<\/sup>M for strept\/avidin-biotin), which explains why the rats in Boas\u2019 original experiments weren’t receiving any biotin. It also suggested quite quickly that this particular interaction, as well as the related interaction between biotin and the\u00a0bacterial<\/a>\u00a0protein streptavidin, could be utilized as a tool in itself.<\/p>\n

For those outside the field, it\u2019s hard to imagine the indirect nature of molecular biology. But let\u2019s have an example. Let\u2019s say you\u2019re studying\u00a0Alzheimer\u2019s disease<\/a>. Now you\u2019ve read up on this disease and you\u2019ve found out that a major protein in this disease is APP (Abeta precursor protein<\/a>), and you\u2019ve hypothesized that (for whatever reason) in diseased people APP gets to the\u00a0cell surface<\/a>\u00a0and is stuck there. What do you do? You decide to grow some\u00a0neurons<\/a>\u00a0on a plate. But the neurons are tiny, you can only see them under a microscope. And APP is even smaller, and you can\u2019t see it. What now? Insofar as you\u2019re concerned, there are heaps of APP proteins in each cell, each of which is moving to various places in the cell, none of which you can see, not even under a microscope. Plus, even if you could see them under a microscope then you\u2019d have to find every single APP protein and see where it went and count them all: impractical. Here\u2019s what you could do using biotinylation (with a cleavable, NHS-biotin):<\/p>\n

You\u2019d pour some biotin over your plates and then leave them for a while. Although you can\u2019t see it, the biotin is sticking to all the proteins on the cell surface. Then you remove the liquid from the cells and wash them a couple of times, and then leave them again. You still can\u2019t see what\u2019s happening, but you know that all the biotin that has stuck to proteins is still there, and all the free biotin is gone. You also know that by leaving them for a while you\u2019re giving the biotinylated APP at the surface an opportunity to go back inside the cell. Now you want to get rid of all the biotin which is still at the surface so that the only biotin left will be that which has gotten inside the cell. You do that by using a special chemical which cuts any biotin at the surface. Now you can explode the cells (with a\u00a0lysis buffer<\/a>), and put the dead cells onto special beads which are coated in streptavidin. Again, you still can\u2019t see what\u2019s happening, but you know that everything that still has biotin attached is getting stuck to the streptavidin, and everything else is floating around. Now by washing the beads, all the non-biotinylated stuff is gotten rid of, so you can boil the beads to free any bound proteins and then do something like a\u00a0Western blot<\/a>\u00a0to quantify how much APP you have.<\/p>\n

Got that? You painted the surface with biotin, then gave the biotin at the surface an opportunity to be taken inside with proteins (including APP), then cut off all remaining surface biotin and killed the cells, and used sticky beads to capture everything that was painted but nothing else. You then used a separate technique for telling you how much APP you had left. The APP you have left is the APP which was at the surface but which got inside in the allotted time. What you can now do is compare cells with Alzheimer\u2019s disease with healthy cells, and see which are better at getting APP back inside the cell. If there\u2019s a difference it might suggest a mechanism for Alzheimer pathology. Phew!<\/p>\n

There are countless applications and variations for biotinylation. Once biotin and streptavidin (and their derivatives) are seen as generic tools, their use is limited only by the researchers’ imagination. Obvious uses include labelling particular cell components or making components that can only travel to particular areas of the cell (e.g. excluded from cross the\u00a0plasma membrane<\/a>).<\/p>\n

But what is it?<\/h3>\n

The rapid expansion of technologies based on the interactions of biotin with its very-high-affinity binding proteins, avidin and streptavidin, in biochemistry, immunology, cell biology, and biotechnology, might obscure the fact that biotin is, in the first place, a vitamin, required by all forms of life. (Smith and Cronan\u00a01999<\/a>)<\/p><\/blockquote>\n

After all that’s been said, it is very easy to forget the fact that biotin is a natural product first and a scientific tool second. It’s worth stating, briefly, what biotin and the avidin proteins are, and how their use relates to their natural function.<\/p>\n

Biotin:<\/h3>\n

Before cells can use biotin, they need to acquire it, and unlike\u00a0bacteria<\/a>,\u00a0higher organisms like humans can\u2019t make their own so depend on biotin in their diet. Because biotin is so important for survival, and because it\u2019s a relatively rare commodity, cells have complex mechanisms for getting biotin and then recycling it.<\/p>\n

Biotinylation is a\u00a0post-translational modification<\/a>. This means that it is something that gets added to the\u00a0protein<\/a>\u00a0after the protein has already been made. Modifications can have different effects. Examples include\u00a0modifications<\/a>\u00a0that provide an energy source for the protein, allowing it to act, while other\u00a0modifications<\/a>\u00a0label the protein for destruction or transportation to a particular subcellular location. As indicated by its name, biotinylation involves biotin being attached to another protein. In\u00a0eukaryotes<\/a>\u00a0(which includes all animals) biotin is attached to proteins by an enzyme called\u00a0holocarboxylase synthase<\/a>.<\/p>\n

Insofar as cell function is concerned, protein biotinylation is important in energy production, in particular in\u00a0fatty acid<\/a>\u00a0synthesis,\u00a0gluconeogenesis<\/a>, and\u00a0amino acid<\/a>\u00a0catabolism<\/a>. In all of these energy-related functions, the biotin is used to receive and then pass on a\u00a0carboxyl<\/a>\u00a0group, in other words, it is used to receive and then pass on a\u00a0carbon dioxide<\/a>. (Interestingly, biotinylation of\u00a0histone<\/a>\u00a0complexes is being explored as an endogenous (eukaryotic) mechanism for regulating\u00a0transcription<\/a>, which to my mind highlights the multidimensional nature of \u201cfunction\u201d in biology).<\/p>\n

Biotin derivatives:<\/h3>\n

The biotin used in the lab is almost never the same biotin which we need in our diet. For starters, the attachment of biotin under experimental conditions is different to that which occurs naturally. Naturally, biotin is attached to very specific proteins by a specific enzyme (holocarboxylase synthase). However, for experimental use, a reactive group is attached to the biotin. For example, by attaching\u00a0N-Hydroxysuccinimide esters<\/a>\u00a0(NHS esters) to the biotin, the NHS ester part attaches to\u00a0amine<\/a>\u00a0groups on proteins by itself, without the need for any enzyme. NHS esters are not the only reactive group that can be used, and others are specific for other parts of proteins or even for\u00a0carbohydrates<\/a>. It\u2019s even possible to use two different reactive groups to allow a single biotin to capture two different targets. Another consideration is the spacer arm. This is the chain of atoms that connect the biotin with the reactive group. The spacer arm can confer different properties onto the biotin molecule. It can affect its\u00a0solubility<\/a>, its ability to cross a cell membrane, it\u2019s length (which may be important to avoid things getting too cluttered), and it\u2019s cleavability (ability to be cut). The spacer arm can be made to be cleavable in the presence of a\u00a0reductant<\/a>\u00a0(e.g. a chemical like\u00a0dithiothreitol<\/a>), or even in the presence of light (i.e. photocleavable). These can be useful for attaching the biotin and then selectively removing it later in an experiment.<\/p>\n

Avidin, streptavidin, and others:<\/h3>\n

Although first found prominently in chicken egg white, avidin is also found in the eggs of other birds, reptiles, and amphibians. It can also be found in some other tissues in those species. Despite avidin being the first protein identified to bind biotin, very little is known about what its natural function is. The best evidence to date is that it plays some\u00a0immune<\/a>\u00a0role: avidin production is increased by tissue trauma as well as bacterial and\u00a0viral<\/a>\u00a0infections. Those facts suggest that avidin might be produced in response to infection or vulnerability to infection, or simply as a\u00a0shield against infection and that it might slow\u00a0pathogens<\/a>\u00a0by binding to them. That would explain why there is so much avidin in egg whites; to protect the developing chick. Having said all that, like everything else in biology, it\u2019s unlikely that avidin serves only one purpose, and there\u2019s research showing avidin to play a role in\u00a0development<\/a>, possibly by regulating\u00a0biosynthesis<\/a>\u00a0to affect\u00a0cell proliferation<\/a>\u00a0in turn. In this context, it seems likely that avidin\u2019s biotin binding capacity is directly involved, and that it binds biotin to regulate biotin\u2019s role in fatty acid metabolism. This is all tentative, but it does suggest that avidin binding to biotin is an endogenous function with specific functional consequences.<\/p>\n

Even compared to the know little about avidin, we know even less about streptavidin. Streptavidin is a protein found in the bacterial species\u00a0Streptomyces avidinii<\/a>, and pretty much all we know about it relates to its extraordinarily strong (non-covalent<\/a>) binding. Nothing seems to be known about what its natural function is, however it is reasonable to presume that the function must relate to its biotin binding capacity. Because streptavidin binds biotin even stronger than avidin, practically all biotin pull-downs are now done using streptavidin.<\/p>\n

Although avidin and streptavidin are the two best-known biotin-binding proteins, other avidin-like proteins from other species have been identified in recent years, including from other bacteria, frogs (xenavidin), and even mushroom (tamavidin). Insofar as biotinylation is concerned, some of the investigators who\u2019ve identified these other biotin-binding proteins have pointed out that the different proteins\u2019 biochemical properties could offer a larger array of tools, as alternatives to streptavidin. Theoretically, that\u2019s true, but to my mind, the main property needed by the binding protein is just binding capacity, other customizations can be achieved more simply by modifying the biotin directly. Having said that, there\u2019s definitely potential for specialized avidin molecules…<\/p>\n

Avidin derivatives:<\/h3>\n

Commercial streptavidin-biotin kits for the time being all depend on modified biotin to confer special properties. However, modified avidin proteins are being generated and explored as offering further specializations to further expand the biotinylation toolkit. For example, changing particular parts of streptavidin can form so-called \u201csmart\u201d streptavidin which can bind biotin per normal, but then releases it under selected conditions. Among other possibilities, this offers an exciting tool for drug delivery. A biotinylated drug can be bound to a smart streptavidin which keeps the drug from acting in the wrong place by only releasing the drug when it gets to a particular environment (for example a\u00a0pH-sensitive smart streptavidin might release the drug when it gets to the appropriate subcellular\u00a0organelle<\/a>). Other possibilities being explored (all of which are still in their early days), is the possibility of forming streptavidin which\u00a0oligomerizes<\/a>\u00a0to form\u00a0signalling platforms<\/a>\u00a0to regulate\u00a0signal transduction<\/a>; using condition-sensitive oligomerizing streptavidin as reporters for changing conditions; for\u00a0enantiomer<\/a>\u00a0selectivity; as well as other possibilities relating to forming streptavidin that are made up of different subunits (natural streptavidin is made up of 4 identical subunits that come together).<\/p>\n

Final thoughts:<\/h3>\n

Trapped in his mortal condition, man refused to bend to the laws of nature.\u00a0Francois Jacob<\/a>\u00a0(Of flies, mice, and men<\/a>)<\/p><\/blockquote>\n

Considered as a molecular tool, biotinylation is an extraordinarily useful and versatile one. But if we take a step back there are a few more\u00a0general<\/a>\u00a0ideas that suggest themselves.<\/p>\n

The first great tool-smith was not the one who realized that a piece of stone could act as an axe, it was the one who took that first sharp tool and used it to create a second one that was even sharper. To my mind, this is technology: tools begetting tools.<\/p>\n

To contemporary biologists, biological systems are not just arenas to be studied, they\u2019re also potential toolkits. The strong binding between biotin and avidins suggested a tool for capturing molecules. Other examples abound: the\u00a0green fluorescent protein<\/a>\u00a0(GFP) from jellyfish is often added to proteins so that they can be seen under the microscope; the heat-resistant\u00a0DNA polymerase<\/a>\u00a0(an enzyme that takes single strands of DNA and copies them)\u00a0Taq<\/a>\u00a0from hot water springs bacteria is routinely used for increasing yields of DNA for purposes of sequencing or cloning, etc etc.<\/p>\n

Taking these two ideas together \u2013 technology as tradition and biology as potential tools \u2013 I\u2019d like to leave off with a philosophical coda.<\/p>\n

Civilization, taken to be progress, is mankind\u2019s desire to recreate the world in hir own image. If we have created ugliness, it is because we\u2019ve seen ourselves as ugly. As we create beauty it is because we know ourselves to be beautiful. The world we find ourselves in is extraordinarily complex and filled with elegant systems. Still, the world wasn\u2019t made for us, and although it displays beauty, it certainly isn\u2019t beauty itself. The world is filled with\u00a0flaws<\/a>. It is broken. To understand the world is to want to recreate it; to take the world and remould it into a new thing, a better thing. We may be an insignificant speck littering a tiny mud-ball drifting around a nondescript star, but we\u2019re also a\u00a0small<\/a>\u00a0point where the universe is self-aware, where the universe sees itself and knows that if it could only know itself it could strive to reincarnate itself as\u00a0perfection<\/a>.<\/p>\n

Major references:<\/b><\/p>\n