What does hemoglobin fingerprinting have to do with crime scene fingerprint fingerprinting? Say “hy” to ninhydrin! From fingerprinting to fossil dating, this chemical helps us see the invisible! Ninhydrin can do this “magic” because it reacts with amines (nitrogen-hydrogen) groups – like those of amino acids (protein letters) – to form a purpley product. And it “doesn’t care” whether those amine groups are located on protein pieces separated on a piece of paper waiting to reveal wondrous insight, sloughed off in a sweaty fingerprint, or buried for thousands of years.
blog form (refreshed from January) : http://bit.ly/ninhydrin
We looked at how Ingram pinpointed the single amino acid (protein letter) difference between normal hemoglobin protein (your blood’s oxygen carrier) and the hemoglobin of sickle cell anemia patients by cutting up the proteins and comparing their pieces. To do this, he developed a “peptide fingerprinting” method to separate the protein fragments (peptides) based on their charge (through electrophoresis in one direction) and solubility in various solvents (through chromatography in the perpendicular direction). more here: http://bit.ly/2tGRlvt
And we saw how Frederick Sanger used a similar strategy when figuring out the sequence of insulin. http://bit.ly/insulinsequencing
When I hear “chromatography” my mind jumps to “colors” – that’s what “chrom-“ means, right? And this always made sense to me because I would think back to those early paper chromatography experiments I did as a kid where you separate the different colored inks in a marker using paper chromatography. The term “chromatography” did in fact come from its original uses separating colored compounds. In particular, Russian botanist Mikhail Tswett in 1903 used it to separate colored plant pigments. Etymology-wise, chromatography means “writing in color” which I think is so poetically beautiful!
But the molecules Ingram & Sanger had to color were “invisible.” So why call it chromatography?
The thing which makes chromatography chromatography is actually the separation part, not the colors part. You have 2 phases – stationary & mobile. In paper chromatography you have a solid stationary phase which is the filter paper in the column and a liquid mobile phase which is the buffer (pH-stabilized salt water) containing the dissolved things you’re trying to separate. You get the mobile phase to flow through. And you separate components based on which phase they’d rather hang out with.
They could separate the peptide pieces and/or amino acids on pieces of filter paper. But unlike when you might have done chromatography in school by dotting various color markers on a piece of paper and sticking them in liquid to separate the colorful dyes in the ink, the peptides Ingram & Sanger were separating were invisible – so they had to think!
Thankfully he didn’t have to think too hard on this part of the puzzle because scientists had known for a while about the magical powers of a chemical called ninhydrin to reveal the location of amino acids & peptides.
Ninhydrin (1,2,3-triketohydrindene hydrate) is the hydrate of indane-1,2,3-trione. Hydrate basically means each ninhydrin molecule is tightly bound to water molecules, even in the “solid” form. And an indane is a 2-fused-ring thing with a 6-carbon (6-C) benzene ring & a 5-C ring. “Trione” tells you there are 3 -(C=O)s on the 5C ring (we call such groups carbonyls). And the middle carbonyl can “tautomerize” between forms where it has 2 hydroxyl (-OH) groups instead.
But don’t get too familiar with this initial structure because later, after reacting with amino acids, these hydroxyl groups will be ditched, replaced by a nitrogen (N) hooked up to another ninhydrin derivative. And this double ninhydrin thing is purple! It’s sometimes called Ruhemann’s purple because it was discovered (by accident) by Siegfried Ruhemann in 1910 – he followed up and found out it could detect amino acids. And people have found a lot of uses for it ever since.
The kind of amino acids biochemists are usually referring to when we talk about amino acids are alpha amino acids (α-amino acids), and they’re the kind used in proteins. Amino acids have an amino group (-NH₂ or -NH₃⁺ depending on the pH) and a carboxyl group (-(C=O)-OH in the carboxylic acid form and -(C=O)-O⁻ in the carboxylate form). “a” refers to the fact that, in α-amino acids, these groups are attached to the same central “alpha” carbon. This is also the same carbon that is attached to unique side chains (aka R groups) that make different protein letters different. more here: http://bit.ly/37Aym43
The purple product forming from them happens in multiple steps, which you can see in the pics, but basically:
- one ninhydrin reacts with an amino acid in a such a way that the ninhydrin gets stuck to the amino acid’s nitrogen through a “Schiff base.”
- that’s really awkward, so the amino acid ditches carbon dioxide CO₂ (decarboxylates)
- Still not very molecularly comfy, so when water comes along, the part of the amino acid that’s still stuck to the first one gets released as an aldehyde version of the amino acid it once was (instead of a carboxylic acid (-(C=O)-OH) it has an aldehyde group (-(C=O)-H).
- this gives you an intermediate where the ninhydrin now has an amine group where it once had those 2 hydroxyls. And, since ninhydrin likes to interact with amines… this intermediate interacts with another ninhydrin molecule to give you the blue-purple thing.
What do I mean by blue-purple? Ruhemann’s purple has a “maximum absorption at 570nm” – which brings me to “why do things look different colors?” – the short answer is that they absorb different wavelengths of light, such as light with a wavelength (distance between wave peaks) of 570 nanometers. We perceive different wavelengths of light as different colors, and when light is absorbed the “leftover” light containing whatever wasn’t stolen can look different. So white light, which contains all colors of the rainbow, looks different when it has rainbow stripes stolen. If you look at a color wheel, the color something appears to your eye is across from the color that that something absorbs. So Ruhemann’s purple, for example, absorbs a green-yellow light most strongly and we see a blue-purple.
But why does it absorb that light? To understand this we need to back up and discuss what light is. Light is “just” little packets of energy called photons traveling in waves and different colors of light have photons with different amounts of energy. The more energy the photons have, the more they want to move, but they all travel at the same linear speed (the speed of light), so instead of traveling faster, more energetic photons take a more slalom-y path, up-down-up-down-ing more along the way. As a result, the more energetic the photon, the higher the frequency of the waves, and the tighter packed the peaks are (shorter the wavelength).
When we think about light, we usually think about “visible light” but that only reflects a small slice of a vast electromagnetic radiation (EMR) spectrum which also includes things like microwaves and infrared on the slow side and ultraviolet (UV) & x-rays on the fast side. Visible light is just the portion of the EMR spectrum that our eyes have receptors for – the least energetic (longest wavelength) light we can see is reddish and the the most energetic (shortest wavelength) light we can see is purplish.
A lot of times, things absorb light of wavelengths that we can’t see. So we don’t realize those wavelengths have been stolen. But if something absorbs visible light, we can see it. So a way to make something invisible visible is to change it so that it absorbs visible light.
Now we can revisit why things absorb different wavelengths of light in the first place. This is where it helps to go back to thinking of light as energy packets instead of waves. Molecules are made up of atoms joined together by sharing negatively-charged subatomic particles called electrons. The electrons whizz all around but there are places they most like to hang out, and we call these “orbitals.” You can think of them kinda like electron housing units and electrons have to “pay rent” in the form of energy to live there. The further away from the central atomic nucleus (containing the positively-charged protons charged with reigning them in), the more energy is required to live there.
If an electron absorbs a photon with the exact right amount of energy to pay the rent difference between their current orbital and a further-out one, they can “get excited” and temporarily move to a higher-energy, further from the nucleus, orbital (and then usually fall back down with the energy released as heat, although in the case of fluorescence, that energy is released as light of a different wavelength). The amount of energy required for the move depends on the atoms involved & how they’re hooked up, so different molecules absorb different wavelengths of light.
The atoms in proteins are hooked up such that they usually absorb ultraviolet light (most strongly at 280 & 230).This is helpful if you have a UV detector (like on a protein-purification chromatography machine that monitors the UV of liquid coming off the column to tell when your protein elutes), but not so helpful if you’re trying to see protein with your eyes because we can’t see UV, so can’t tell whether any light has been stolen.
Instead, if we want to see evidence of protein, we need something that absorbs lower-energy light (light with a longer wavelength, something in the visible spectrum). And for something to absorb lower-energy light it needs to have closer-together orbitals to move between. Great places to find these more crowded housing arrangements are “aromatic” molecules.
Aromaticity isn’t about smelliness – instead it’s about ring-y “resonance.” Atoms link up by sharing pairs of electrons – you need 2 for a single bond & 4 for one of the shorter, stronger, double bonds. But sometimes groups of atoms have more than enough electrons for single bonds but don’t have quite enough to double bond them all, so they form a sort of electron commune known as resonance/electron delocalization/conjugation in which the “extra” electrons are shared evenly among the group. And when atoms do this in a ring form, we call them aromatic. more here: http://bit.ly/2qzMRFi
Basically, after they’ve “spent” 1 electron each on the bonds to their neighbors they donate their “extra” into a communal shared stock. Those atoms that opt into this commune get to share, and this leads to electron delocalization above and below aromatic rings, kinda like a donut. This electron delocalization through resonance involves the merging of some neighboring molecules’s orbits into that shared donut. And this lowers the cost to move to promote an electron, so an electron-conjugated molecule can absorb lower-energy (longer-wavelength) light.
Reacted-ninhydrin, Ruhemannn’s purple, is one of those really-aromatic molecules. So when ninhydrin reacts with amino acids and peptides, you can see a colored product.
Ninhydrin works well for amino acids and peptides, but not so well for whole proteins. There are a couple of reasons for this. When you have free amino acids, each one has at least one amino group (from the generic part). But when amino acids link up to form proteins, that amino group gets used in the linkage – it joins the carbonyl carbon of another amino acid, turning it into an amide (N next to a (C=O)) which ninhydrin doesn’t like. So the only free backbone amino group in a protein or peptide is at one end (the N-terminus). There are also primary amine groups scattered around the protein in the lysine residues. But in the context of full-on proteins, steric hindrance (the molecular version of “stay out of my personal space”) prevents them from reacting readily with the ninhydrin. With short peptides, you have a much higher concentration of ends than with full proteins, and even more ends with free amino acids. So lots of primary amine groups – most of the time… [gives proline the side-eye]
First, what do I mean my primary amine? “Amine” is a name we give to nitrogen attached to things and we can further classify amines as primary (attached through a single bond to a single carbon-containing group (abbreviated C)), secondary (2 links to carbon), tertiary (3 links), or quaternary (4 links to C). And to get Ruhemann’s purple you need a primary amine. For most amino acids this isn’t a problem – free amino acids usually have a primary amine at their end (a nitrogen attached to only 1 carbon). But proline’s weird. It has a secondary amine (nitrogen attached to 2 carbons) because its side chain curves back on itself and binds the nitrogen in the backbone. When it reacts with ninhydrin, it can’t form a Schiff base because it doesn’t have the hydrogen needed, so the ninhydrin gets stuck, giving you an “iminium salt” – it’s also colored, but its more yellow-orange (max absorption at 440nm)
But a cool thing about ninhydrin and most amino acids is that the colored product gets released from the amino acid. And the same colored product regardless of which one because the only amino-acid-derived part of the colored product is the nitrogen.
As I mentioned before, Ruhemann discovered ninhydrin’s magical amino acid revelatory power in 1910, and it quickly became widely used – in addition to people like Ingram & Sanger using it to develop their chromatograms, scientists used it to measure amino acid quantities based on “how blue/purple” solutions became when you added it.
There have been a lot of biochemical uses for it, but there are also some really random ways ninhydrin is used…
Like in forensics, where it’s used to develop fingerprints. It wasn’t used in this way until the mid-1950s (despite scientists knowing for years that it reacts with sweat and thus advising each other to avoid touching anything they want to test with it (no fingerprints on my chromatogram!)). But it quickly found widespread use by CSI folks – and it works because sweat contains, among other things like water (which is 98% of sweat), oils, salts, etc, a small amount of amino acids (with serine being the major one) that stay behind when sweat evaporates and is no longer wet. It’s only a tiny amount (~250ng per fingerprint) but it’s enough for ninhydrin to detect. In addition to the classical ninhydrin, there have been versions made that are more stable, etc. https://www.ncjrs.gov/pdffiles1/nij/225327.pdf
The crime scene fingerprinting thing takes advantage of amino acids in sweat to see fingerprints – there’s also a medical test which takes advantage amino acids in sweat to see sweat – or lack of it, which can indicate nerve damage. To test for laceration of the nerves innervating a patient’s fingertips, a doctor places their fingertips on bond paper and traces them. Then the paper is sprayed with ninhydrin stray and fixer reagent – areas of sweat appear as dots – if there has been a complete nerve laceration, that nerve’s innervation area won’t have any dots.
Another medical usage of ninhydrin is the “Hippurate test” – this lab test is used to help diagnose certain bacterial infections – cultures of the patient’s blood are mixed with a chemical called hippurate. Some (but not all) types of bacteria make an enzyme called hippuricase, which is able to cleave that hippurate into benzoic acid & the amino acid glycine. And then ninhydrin is added to detect the glycine. A positive result gives a blue-purple color and can indicate the presence of types of bacteria including Group B streptococci (e.g. Streptococcus aglactiae), Campylobacter jejuni, Listeria monocytogenes, or other hippuricase-makers. So it’s just one in a toolbox of tests used for bacterial IDs.
How about avoiding medical complications in the first place? A ninhydrin test can be used to detect residual protein parts on re-usable surgical instruments – the instruments are “swabbed” with water-wetted rayon swabs, then those swabs are dipped in ninhydrin & heated to ~60° for up to one hour. If it turns purple, the instrument should be rewashed.
And what about that fossil-dating I alluded to? When ninhydrin reacts with free alpha amino acids, carbon dioxide is released. So when ninhydrin reacts with amino acids from the collagen from fossil bones, CO₂, is released. And the carbon of that CO₂ was from when the protein was made. So scientists can use it to “carbon date” the fossil using through a type of radiocarbon dating called gas proportional counting – basically carbon-14 is an unstable weakly-radioactive version of carbon that is produced constantly in the atmosphere when nitrogen-14 atoms get bombarded by cosmic ray neutrons. And it comes to Earth as a tiny amount of “heavy” CO₂ that plants breath in and assimilate into proteins, etc. over their lifetime. And animals eat and use it to make their own proteins, etc. And they also break down things they. made from it, exchanging the CO₂ back with the atmosphere, so there’s a fairly constant ratio of normal carbon (C-12) (which is almost all of it) and carbon-14 in the animal throughout its life. But when something dies, it stops exchanging CO₂, so they can’t assimilate any more of it, but any of the “heavy” carbon-14 in their proteins, etc. is stuck there. But it starts decaying, and giving off detectable radiation as it does so. And scientists can measure this to figure out how old something is based on how much C-14 vs normal there is now. more on radioactivity: http://bit.ly/2m5sGME