Nobel Laureates: Max Perutz and John Kendrew

 
 
The Nobel Prize in Chemistry 1962.

"for their studies of the structures of globular proteins"


Max Perutz (1914-2002) and John Kendrew (1917-1997) won the Nobel Prize in 1962 for solving the structures of hemoglobin (Perutz) and myoglobin (Kendrew). This is the same year that Watson, Crick, and Wilkins won for the structure of DNA [Nobel Laureates: Francis Crick, James Watson, and Maurice Wilkins]. Recall that Watson & Crick were working in the Perutz lab at the time of their discovery and Crick was actually working on the structure of hemoglobin as part of his Ph.D. thesis [The Story of DNA (Part 1)].

1962 was also the year that John Steinbeck won the prize for literature [see Nobel Laureates 1962].

The Presentation Speech was given by Professor G. Hägg, member of the Nobel Committee for Chemistry of the Royal Swedish Academy of Sciences. Those of you who weren't yet born in 1955 should make note of the fact that this work required an enormous number of calculations that were only made possible with the help of "a very large electronic computer." Many of my students are surprised to discover that biochemists have been working with computers for over fifty years. Most of them think that computers weren't invented until about 1990 when they were just babies.

Your Majesties, Your Royal Highnesses, Ladies and Gentlemen.

In the year 1869 the Swedish chemist Christian Wilhelm Blomstrand wrote, in his at that time remarkable book Die Chemie der Jetztzeit (Chemistry of Today):

"It is the important task of the chemist to reproduce faithfully in his own way the elaborate constructions which we call chemical compounds, in the erection of which the atoms serve as building stones, and to determine the number and relative positions of the points of attack at which any atom attaches itself to any other; in short, to determine the distribution of the atoms in space."

In other words, Blomstrand gives here as his goal the knowledge of how compounds are built up from atoms, i.e. knowledge of what is nowadays often called their "structure". Moreover, structure determination has been one of the biggest tasks of chemical research, and has been approached using many different techniques. For several reasons, the structure determination of carbon compounds, the so-called organic compounds, experienced an initial rapid development. At this stage the techniques were generally those of pure chemistry. One drew conclusions from the reactions of a compound, one studied its degradation products, and tried to synthesize it by combining simpler compounds. The structure thus arrived at, however, was in general rather schematic in character; it showed which atoms were bonded to a given atom, but gave no precise values for interatomic distances or interbond angles. However, for an up-to-date treatment of the chemical bond and in order to derive a correlation between structure and properties, these values are needed, and they can only be obtained using the techniques of physics.

The physical method which, more than any other, has contributed to our present-day knowledge of these mutual dispositions of the atoms is founded on the phenomenon which occurs when an X-ray beam meets a crystal. This phenomenon, called diffraction, results in the crystal sending out beams of X-rays in certain directions. These beams are described as reflections. The directions and intensities of such reflections depend on the type and distribution of the atoms within the crystal, and can therefore be used for structure determination. It is 50 years ago this year since Max von Laue discovered the diffraction of X-rays by crystals, a discovery for which he was awarded the 1914 Nobel Prize for Physics. This work opened up a whole new range of possibilities for studying both the nature of X-rays and the structure of compounds in the solid state. The initial application of structure determination was developed first and foremost by the two English scientists, Bragg father and son, and as early as 1915 they were rewarded with the Nobel Prize for Physics. The techniques have since been considerably refined, and it has been possible to solve more and more complicated structures. However, considerable difficulties were encountered as soon as any other than very simple structures were considered. There is no simple general way of progressing from experimental data to the structure of the compound under investigation. Moreover, the mathematical calculations are exceedingly time-consuming. However, by about the middle of the 1940's a point had been reached where it was becoming possible to carry out X-ray determinations of the structures of organic compounds which were so complicated that they defied all attempts using classical chemical methods.

In 1937 Max Perutz performed some experiments in Cambridge to find out whether it might be possible to determine the structure of haemoglobin by X-ray diffraction, since no other method could be imagined for this purpose. Sir Lawrence Bragg, who tirelessly continued the work begun jointly with his father, in 1938 became the head of the Cavendish Laboratory in Cambridge. When he saw the results obtained by Perutz, he encouraged him to continue and has ever since lent a very efficient support. Haemoglobin belongs to the proteins which play such an enormous part in life processes, and which are a basic material in living organisms. Haemoglobin is a component of the red blood corpuscles. It contains iron which can take up oxygen in the lungs and later give it up to the body's other tissues. Haemoglobin is counted among the globular proteins, whose molecules are nearly spherical. It was chosen for the initial attempt, partly because it could develop good crystals, and partly because the haemoglobin molecule is quite small for a protein molecule. About ten years later, John Kendrew joined Perutz' research group, and the task allotted to him was to try to determine the structure of myoglobin. Myoglobin is another globular protein, closely related to haemoglobin, but with a molecule only a quarter as large. It is found in the muscles, and enables oxygen to be stored there. Particularly large amounts of myoglobin are found in the muscular tissues of whales and seals, which need to be able to store large quantities of oxygen when diving.

However, Perutz and Kendrew encountered considerable difficulties. In spite of exceptionally comprehensive work, the result was not forthcoming until 1953, when Perutz succeeded in incorporating heavy atoms, namely those of mercury, into definite positions in the haemoglobin molecule. By this means the diffraction pattern is altered to some extent, and the changes can be utilized in a more direct structure determination. The method was already known in principle, but Perutz applied it in a new way, and with great skill. Kendrew also succeeded, by an alternative method, in incorporating heavy atoms, generally mercury or gold, into the myoglobin molecule, and could subsequently proceed in an analogous manner.

A necessary condition for this technique is that the addition of the heavy atoms should not alter the positions of the other atoms of the molecule within the crystal. In this connection it is simply because of its enormous dimensions that the molecule remains practically unaltered. Bragg has rather aptly said that "the molecule takes no more notice of such an insignificant attachment than a maharaja's elephant would of the gold star painted on its forehead".

But even if the path was now open for a direct structure determination of haemoglobin and myoglobin, there was still an enormous amount of data to be processed. Myoglobin, the smaller of the two molecules, contains about 2,600 atoms, and the positions of most of these are now known. But for this purpose, Kendrew had to examine 110 crystals and measure the intensities of about 250,000 X-ray reflections. The calculations would not have been practicable if he had not had access to a very large electronic computer. The haemoglobin molecule is four times as large, and its structure is known less thoroughly. In both cases, however, Kendrew and Perutz are currently collecting and processing an even greater number of reflections in order to obtain a more detailed picture.

As a result of Kendrew's and Perutz' contributions it is thus becoming possible to see the principles behind the construction of globular proteins. The goal has been reached after twenty-five years' labour, and initially with only modest results. We therefore admire the two scientists not only for the ingenuity and skill with which they have carried out their work, but also for their patience and perseverance, which have overcome the difficulties which initially seemed insuperable. We now know that the structure of proteins can be determined, and it is certain that a number of new determinations will soon be carried out, perhaps chicfly following the lines which Perutz and Kendrew have indicated. It is fairly certain that the knowledge which will thus be gained of these substances which are so essential to living organisms will mean a big step forward in the understanding of life processes. It is thus abundantly clear that this year's prize-winners in chemistry have fulfilled the condition which Alfred Nobel laid down in his will, they have conferred the greatest benefit on mankind.

Doctor Kendrew and Doctor Perutz. One of you recently said that today's students of the living organism do indeed stand on the threshold of a new world. You have both contributed very efficiently to the opening of the door to this new world and you have been among the first to obtain a glimpse of it. Through your combined efforts there is now in view, as it has been stated by yourself, a firm basis for an understanding of the enormous complexities of structure, of biogenesis and of the functions of living organisms both in health and disease.

It is with great satisfaction, therefore, that the Royal Swedish Academy of Sciences has decided to award you this year's Nobel Prize for Chemistry for your brilliant achievement.

On behalf of the Academy I wish to extend to you our heartiest congratulations, and now ask you to receive from the hands of His Majesty the King the Nobel Prize for Chemistry for the year 1962.

The figures are taken from A Little Ancient History by Richard (Dick) Dickerson. The top figure shows the myoglobin/hemoglobin group outside the New Cavendish Laboratory in 1958. That's Maz Perutz in the white lab coat. The second picture is a remarkable photograph of two postdocs, Bror Strandberg (back) and Dick Dickerson (front) carrying the paper tapes for the myoglobin 2A data set. They are just outside the EDSAC II computing centre.

Hemoglobin

 
The following text is slightly modified from Horton et al. (2007) Principles of Biochemistry.

In vertebrates, O₂ is bound to molecules of hemoglobin for transport in red blood cells, or erythrocytes. Viewed under a microscope, a mature mammalian erythrocyte is a biconcave disk that lacks a nucleus or other internal membrane-enclosed compartments (right). A typical human erythrocyte is filled with
approximately 3 × 10⁸ hemoglobin molecules.

Hemoglobin is more complex than myoglobin because it is a multisubunit protein. In adult mammals, hemoglobin contains two different globin subunits called α-globin and β-globin. Hemoglobin is an α₂β₂ tetramer, which indicates that it contains two α chains and two β chains. Each of these globin subunits is similar in structure and sequence to myoglobin, reflecting their evolution from a common ancestral globin gene in primitive chordates.


Each of the four globin chains contains a heme prosthetic group identical to that found in myoglobin. The α and β chains face each other across a central cavity (above). The tertiary structure of each of the four chains is almost identical to that of myoglobin (left). The α chain has seven helices, and the β chain has eight. (Two short α helices found in β-globin and myoglobin are fused into one larger one in α-globin.) Hemoglobin, however, is not simply a tetramer of myoglobin molecules. Each α chain interacts extensively with a β chain, so hemoglobin is actually a dimer of αβ subunits. The presence of multiple subunits is responsible for oxygen-binding properties that are not possible with single-chain myoglobin.

The structure of hemoglobin was solved by Max Perutz [Nobel Laureates].



©Laurence A. Moran and Pearson Prentice Hall 2007

Myoglobin

 
Myoglobin is the simplest type of oxygen carrying molecule in vertebrates. It consists of a single polypeptide chain bound to a heme group. The example shown on the left is sperm whale myoglobin. It shows the heme group edge on (gray) bound to a molecule of oxygen (red balls in the middle of the heme group on the right). The other oxygens, left and top, are part of the heme molecule.

The oxygen is bound to the iron atom at the center of the heme group. In the absence of oxygen this iron atom interacts with the side chains of two histidine residues in the myoglobin polypeptide chain. When oxygen binds it forms a bridge between one of the histidine residues (His-64) and the iron atom in the heme group.

Although the oxygen molecule is tightly bound in this configuration it is still capable of being released under the right conditions. Those conditions can be found inside cells that have become depleted in oxygen. Myoglobin is usually found in muscle cells in vertebrates where it plays a role in storing oxygen. The structure of myoglobin was determined by John Kendrew [Nobel Laureates].

Myoglobin is a member of a large family of globins. They include hemoglobin and similar oxygen carrying molecules in bacteria, plants, and other animals. The myoglobins have evolved from ancestral globins to specialize in oxygen storage inside cells.

©Laurence A. Moran and Pearson Prentice Hall 2007

Heme Groups

 
Monday's Molecule #37 is the heme group found in myoglobin and hemoglobin. The heme group consists of a ring structure, called a tetrapyrrole ring system, complexed to a central iron atom. There are many different kinds of these tetrapyrrole structures in cells. They are distinguished by slight changes in the chemistry of the ring system. This particular structure (left) is called protoporphyrin IX. The structure was originally determined by Hans Fischer [Nobel Laureate: Hans Fischer],

The red color of blood is due to the presence of the heme group, which absorbs visible light. Note that the pyrrole rings are linked by methene bridges (-CH=) to create a conjugated double bond system where electrons can be shared all across the ring. Not only does this mean that these rings can absorb photons, it also means that they can accommodate additional electrons without too much trouble.

This is why there are many heme proteins that are involved in oxidation-reduction reactions (reactions that transfer electrons from one substrate to another). For example, cytochrome c has a similar kind of heme group (right). Cytochrome c is a major player in membrane associated electron transport systems in bacteria and mitochondria and in photosynthesis.

Heme type molecules are always tightly bound to proteins. Such molecules are called prosthetic groups and there are two types. The heme in hemoglobin is bound by many weak interactions such as hydrogen bonds and van der Waals interactions. The heme in cytochrome c is an example of a covalently bound prosthetic group. It is attached to its protein by bonds between the edge of the porphyrin ring and cysteine (Cys) side chains in the protein.

Chlorophyll (left) is another type of tetrapyrrole ring molecule but it differs from most others because the central chelated metal ion is magnesium (Mg) instead of iron. Chlorophyll molecules absorb light very efficiently and that's why they play such an important role in photosynthesis. Photosynthesizing organisms—bacteria, algae, plants—have dozens (or hundreds) of chlorohyll molecules packed in their membranes.


©Laurence A. Moran and Pearson Prentice Hall 2007

Physicians Are "Science Professionals"

 
At least that's what the IDiots say [Medical Doctors a Fast Growing Segment of Darwin Doubting Science Professionals].

Who knew? I suppose we shouldn't be surprised if they think an M.D. degree makes you a "science professional." After all, these are some of the same people who think the Earth is only 10,000 years old.

UPDATE: Turns out that many of these medical doctors are actually dentists [Dentists Against Darwin]. Sheesh!

Your View of Evolution

 
The poll for August asks you to identify the person who comes closest to representing your view of evolution. Check out the left hand margin.

70% of Sandwalk Readers are Atheists

 
According to the latest poll (see left hand margin) 70% of Sandwalk readers are atheists (PZ would be proud.). 12% are agnostics—I guess Wilkins and Catshark figured out how to vote multiple times. Only 14% are believers. I wish there were more believers, it would make for more lively discussions.

Six of you are uncertain. Why?

Another Bad Review of The Edge of Evolution

PZ Myers draws attention to another review of Michael Behe's new book The Edge of Evolution [Behe gets another thumbs-down]. This review is published in the July issue Discover magazine [The Simplistic Manifesto]. The author is Cory S. Powell.

I disagree with PZ. This is not a good review. Actually, it's a very bad review. Like many of the published reviews of The Edge of Evolution the author seems to have been reading a far different book than the one I read. Powell says,

To reach this conclusion, Behe makes a number of invalid assumptions about how molecules evolve and interact. He alleges that, because many functional adaptations require multiple changes in proteins, two or more mutations must occur together at the same time in the same gene and only rarely can several mutations "sequentially add to each other to improve an organism’s chances of survival." But in fact natural selection does work on transitional forms, as molecules and traits evolve stepwise. Stepwise evolution has been well documented; one good instance of this is the emergence of color vision. Mutations add up little by little, leading to major changes to proteins over time.

The essence of Behe's argument is not that it's impossible to evolve a double mutation if each one is beneficial. The whole point of the book is that stepwise evolution requires that each step is beneficial. The evidence, according to Behe, shows that many cases involving double mutations involve intermediates that are disadvantagous. Thus, the double mutant had to arise in a single step and this is highly unlikely.

Behe isn't always as clear as he should be but he does make it perfectly clear that he accepts the mechanism that Powell describes. Thus, Powells' criticism is inappropriate and this makes it a bad review. Apparently Powell didn't read the section on the evolution of antifreeze proteins in fish (pp. 77-81) where Behe describes each of the many steps that lead to the modern antifreeze proteins.

Each step would have given the fish some protection against freezing water. Thus, Behe concludes,

Even though we haven't directly observed it, the scenario seems pretty convincing as an example of Darwinian evolution by natural selection. It's convincing because each of the steps is tiny&mdash'no bigger than the step that yielded the sickle cell mutation n humans—and each step is an improvement.

The Discovery review points out that complex combinations of mutations can arise in a stepwise manner by standard Darwinian mechanisms. It implies that Behe never thought of this in his book but that's total nonsense. Of course he did. Behe doesn't deny that phenotypes requiring multiple steps can be produced by random mutations, as long as each step is beneficial. The essence of his argument is that it's impossible to generate phenotypes that require multiple random mutations if the intermediates aren't beneficial.

I'm not arguing that Behe is correct. In fact, I'm preparing a series of postings that will challenge some of his ideas. What I'm objecting to is the mischaracterization of Behe's arguments in many of the published reviews. If you're going to criticize Behe then challenge the argument he makes in the book; namely, that most stepwise pathways are impossible because the intermediates are less fit than their parents.

Powell makes another common mistake in his review. He says,

Behe makes another big, related error in the way he interprets how proteins work together. He contends that for even three proteins to evolve in a cooperative association is wildly improbable, "beyond the edge of evolution." Within a protein, five or six amino acids (components of proteins) need to change simultaneously for it to bond with another protein, according to Behe. From this he concludes that it is impossible for proteins’ interaction to evolve, again requiring life to have been programmed for success from the start. Plenty of evidence contradicts this assertion, however. Many proteins within cells interact with other proteins in ways in which only two or three amino acids are critical for binding.

Behe admits that you may only need three or four selected changes in order to generate a new binding site (p. 114). He agrees that the evolution of a single binding site is within reach of evolution but the simultaneous generation of two binding sites is beyond the edge of evolution because the probabilities are so low. The point is that there are many complexes that require the interaction of several different proteins and the intermediates—where only two proteins interact—are not beneficial. Refuting Behe's real arguments requires a little more effort than the superficial criticism of arguments that Behe is not making.

Powell continues,

Such simple binding sites can arise frequently in proteins. And such interactions form the networks that regulate all sorts of physiological processes in cells and organisms. Cell biologists and biochemists are increasingly finding that, in truth, protein interactions and networks are easy to evolve. Behe should know this—but he has a long history of alleging evolutionary impossibilities and ignoring the scientific literature.

Powell is completely missing the point here. Behe does not deny that such complexes exist, nor does he deny that they evolved in the sense that they arose in organisms whose ancestors didn't have them. Once the mutations occurred, they became fixed in the population by natural selection. Furthermore, Behe does not deny that these networks are "easy to evolve." In fact, they are so "easy to evolve" that they cannot be explained by natural selection acting on random mutations as "Darwinism" requires. Thus, mutations cannot be random.

You don't refute Behe by pointing to examples of evolution by common descent or natural selection; this includes evolution of protein complexes. That's not the point. The point is that "Darwinian" evolution, according to Behe, must require small steps where each step is beneficial and this cannot be demonstrated. Indeed, in many cases the intermediates will likely be detrimental. The conclusion is that multiple mutations have to occur simultaneously as in some drug resistance. For most populations the probability of this happening by random mutation is very small. The fact that it happened is evidence of directed mutation, or so Behe thinks.

In order to show that Behe is wrong you have to demonstrate that his understanding of evolution (i.e., "Darwinism") is wrong and this has led him to false conclusions about probabilities. Many reviewers have failed to do this, possibly because they accept Behe's version of Darwinism.

You can read Michael Behe's responses to his reviewers on the Amazon.com site [Michael Behe's Amazon Blog]. I think it's fair to say that Behe makes some good points (and many bad ones) when he accuses his reviewers of misrepresentation.

Virtual Toronto

 
Here's a site that combines an interactive map of Toronto with images from selected streets [Toronto Virtual City]. The photograph (left) shows the entrance to my building on the University of Toronto campus. The view is looking north from College St.

The satellite view is about two years old. It was taken when the new building was still under construction so the street level image doesn't match the satellite view.

Hmmm ... that reminds me. How come we don't see any more postings where we have to identify a university campus? I forgot which blog that was on.

[Hat Tip: Monado]

Gene Genie #12

 


Gene Genie #12 has been posted at My Biotech Life [Gene Genie #12 aka The Dozen].

The image is from the article on snpedia [WikiPedia Meets Genetics]. Read about how you can access the personal genome of Jim Watson and Craig Ventor.

Monday's Molecule #37

 
Today's molecule looks complicated but it has a very simple name. The short common name of this molecule is not sufficient—you have to supply the correct biochemical name that distinguishes this molecule from similar ones found inside all cells. You're more than welcome to supply the complete IUPAC name if you know it.

There's an indirect connection between this Monday's Molecule and Wednesday's Nobel Laureate(s).

The reward (free lunch) goes to the person who correctly identifies the molecule and the Nobel Laureate(s). Previous free lunch winners are ineligible for one month from the time they first collected the prize. There's only one (Marc) ineligible candidates for this Wednesday's reward since many recent winners haven't collected their prize. The prize is a free lunch at the Faculty Club.

Comments will be blocked for 24 hours. Comments are now open.

Canadian Dinosaur Coins

 
The Royal Canadian Mint has dinosaur coins for sale. Here's a picture of the first one. It shows a fossil Parasaurolophus, a crested, duck-billed species from Alberta.

Future coins will depict Triceratops (2009); Tyrannosaurus rex (2009); and Dromaeosaurus (2010). The face value of the coins is $4 (CDN)—that's currently about $3.80 in US currency but it will be about $4.60 by the time the last coin is issued unless the US dollar stops falling. (Getting out of Iraq would help.)

Only 20,000 coins are being minted. The finish on the "fossil" image is impossible to reproduce exactly so each coin will be slightly different in tone and color. You can buy them for $39.95 (CDN).

How many people know what "D.G. Regina" stands for? (Hint: it's not an atheist slogan.)

The OUT Campaign

 
RichardDawkin.net has started something called the The OUT Campaign. The goal is to encourage all non-believers (atheists) to come out of the closet and make their rejection of religious superstition known. You're supposed to use the red "A" as a symbol to declare that you are an atheist. Several bloggers have put it on their website.

I do not believe in God. I am an atheist. However, the fact that I don't believe in something is often the only thing I have in common with other atheists. It seems a bit silly to form a club based only on what you don't believe in. It would be like having a club for everyone who doesn't believe in Bigfoot, or Santa Claus.

So, while I am happy to announce my preference for rationalism over superstition and proud to be an atheist. I won't be joining any organization based on a negative. I am a proud member of Skeptics Canada and The Centre for Inquiry, Toronto because they stand for something positive.

[Hat Tip: PZ Myers]

The Aliens Are Coming

 
Friday's Urban Legend: False

			The following email message is going the rounds. ALIENS ARE COMING TO ABDUCT ALL THE GOOD LOOKING AND SEXY PEOPLE. YOU WILL BE SAFE, I'M JUST EMAILING TO SAY GOODBYE. We know it's false because I'm still here.

Theme: Deoxyribonucleic Acid (DNA)

 
THEME

Deoxyribonucleic Acid (DNA)

1. Wellcome Trust Images


2. A Strange Molecule


3. Monday's Molecule #35 (ethidium)


4. DNA Is a Polynucleotide


5. Tautomers of Adenine, Cytosine, Guanine, and Thymine


6. Nucleotides Can Adopt Many Different Conformations


7. Nobel Laureates: Francis Crick, James Watson, and Maurice Wilkins


8. The Chemical Structure of Double-Stranded DNA


9. The Three-Dimensional Structure of DNA


10. The Story of DNA (Part 1) Where Rosalind Franklin Teaches Jim and Francis Something about Basic Chemistry


11. Ethidium Bromide Binds to DNA


12. Rosalind Franklin Announces the Death of the Helix


13. Nobel Laureates 1962


14. The Story of DNA (Part 2)Where Jim and Francis Discover the Secret of Life


15. DNA With Parallel Strands


16. Measuring Stacking Interactions


17. Are You as Smart as a Third Year University Student?


18. Rosalind Franklin's Birthday


19. The Watson & Crick Nature Paper (1953)


20. The Franklin & Gosling Nature Paper (1953)


21. The Wilkins, Stokes and Wilson Nature paper (1953)


22. Ethidium Bromide Is a Dangerous Chemical


23. Jim Watson on the Discovery of the Double Helix


24. DNA Tatoo


25. DNA Polymerase I and the Synthesis of Okazaki Fragments


26. Play the DNA Double Helix Game

The Wilkins, Stokes and Wilson Nature paper (1953)

Wilkins published his work on the structure of DNA in the same issue of Nature as the Watson & Crick paper and the Franklin & Gosling paper. The coauthors on the wilkins paper were A.R. Stokes and H.R. Wilson. They were reunited in 1993 on the 40th anniversary of the publication as shown in the photo. (From left to right: Raymond Gosling, Herbert Wilson, Maurice Wilkins and Alec Stokes.)

A copy of the Wilkins, Stokes and Wilson paper is here.

The title of the paper "Molecular Structure of Deoxypentose Nucleic Acids" indicates that this is a paper that will discuss details and experimental results. This is a paper that emphasizes the similarities between X-ray diffraction patterns of DNA fibres from calf thymus, wheat germ, herring sperm, human, and T₂ bacteriophage. They also look at DNA in vivo by examining intact sperm heads, bacteriophage, and animal viruses. The authors conclude that all these DNA have the same general structure and that it is consistent with the model proposed by Watson & Crick.

The Franklin & Gosling Nature paper (1953)

Rosalind Franklin and Raymond Gosling published their results on the structure of DNA in a Nature paper that immediately followed the famous Watson & Crick paper [The Watson & Crick Nature Paper (1953)]. Franklin had completed the manuscript before traveling up to Cambridge to see the Watson & Crick model of DNA but she was able to make changes to her paper before submitting it in early April 1953. A PDF of the paper as it appeared in the journal is here and the original manuscript is here.

The title of the paper, "Molecular Configuration in Sodium Thymonucleate," gives us a clue to why this paper has been ignored and the Watson & Crick paper gets all the attention. The Franklin & Gosling paper is full of obscure references and equations and it's significance can only be recognized because of the paper that preceded it in the April 25th, 1953 issue of Nature. The writing style is ponderous and it does not convey any of the sense of excitement found in the Watson & Crick paper [see April 25, 1953: Three papers, three Lessons].

Franklin and Gosling conclude that DNA is "probably helical," the phosphate groups lie on the outside, and there are probably two strands. They state,

Thus our general ideas are not inconsistent with the model proposed by Watson and Crick in the preceding communication.

As is the case in the Watson & Crick paper, papers in the same issue of the journal are not specifically referenced. If you follow the link to the typed manuscript (above) you can see that this sentence was inserted by hand.

Franklin & Gosling acknowledge their colleagues at the end of the paper in the same manner we saw in the Watson & Crick paper.

We are grateful to Prof. J.T. Randall for his interest and to Drs. F.H.C. Crick, A.R. Stokes, and M.H.F. Wilkins for discussion.

The Watson & Crick Nature Paper (1953)

Watson & Crick submitted their paper on the structure of DNA to the journal Nature on April 2, 1953. It was published in the April 25th issue—a remarkably rapid publication even for that time. A PDF of the paper as it appeared in the journal is here. The original typed manuscript is here.

Now that we've learned about the structure of DNA and it's history [Theme: DNA] we're in a position to work through this seminal paper line-by-line. Let's begin with the title and the opening sentence.

A Structure for Deoxyribose Nucleic Acid

We wish to suggest a structure for the salt of deoxyribose nucleic acid (D.N.A.). This structure has novel features which are of considerable biological interest.

The name of this important molecule is now deoxyribonucleic acid but in 1953 there was no standard nomenclature so Watson & Crick used a common name.

The first sentence is a classic understatement and you can be sure that it's written by Crick and not Watson.

A structure for nucleic acid has already been proposed by Pauling and Corey¹. They kindly made their manuscript available to us in advance of publication. Their model consists of three intertwined chains, with the phosphates near the fibre axis, and the bases on the outside. In our opinion, this structure is unsatisfactory for two reasons:

(1) We believe that the material which gives the X-ray diagrams is the salt, not the free acid. Without the acidic hydrogen atoms it is not clear what forces would hold the structure together, especially as the negatively charged phosphates near the axis will repel each other.

(2) Some of the van der Waals distances appear to be too small.

At the time they wrote the paper, Pauling had not seen their model so Watson & Crick were not certain that he would agree with them. (See Linus Pauling's notes taken during the meeting with Watson & Crick on April 8, 1953.) They were obliged to insert some commentary about competing ideas concerning the structure of DNA, especially the Pauling & Cory model that had just been published several weeks earlier in the Proceedings of the National Academy of Sciences (USA) [Pauling & Cory, 1953]. (The three-stranded structure of DNA from the Pauling & Cory paper is shown above.)

No doubt Watson & Crick were delighted to be able to correct the famous Linus Pauling. The idea that Pauling might have got the structure wrong because of simple mistakes like packing charged molecules together and not allowing for proper van der Waals distances was too tempting to omit.

Another three-chain structure has also been suggested by Fraser (in the press). In his model the phosphates are on the outside and the bases on the inside, linked together by hydrogen bonds. This structure as described is rather ill-defined, and for this reason we shall not comment on it.

Bruce Fraser published a brief note where he took issue with the Pauling & Cory paper but, as Watson and Crick note, the proposed structure is not described in any detail. There are no figures. The Fraser manuscript is here].

We wish to put forward a radically different structure for the salt of deoxyribose nucleic acid. This structure has two helical chains each coiled round the same axis (see diagram). We have made the usual chemical assumptions, namely, that each chain consists of phosphate diester groups joining beta-D-deoxyribofuranose residues with 3',5' linkages. The two chains (but not their bases) are related by a dyad perpendicular to the fibre axis. Both chains follow right-handed helices, but owing to the dyad the sequences of the atoms in the two chains run in opposite directions. Each chain loosely resembles Furberg's² model No. 1; that is, the bases are on the inside of the helix and the phosphates on the outside. The configuration of the sugar and the atoms near it is close to Furberg's "standard configuration," the sugar being roughly perpendicular to the attached base. There is a residue on each every 3.4 A. in the z-direction. We have assumed an angle of 36° between adjacent residues in the same chain, so that the structure repeats after 10 residues on each chain, that is, after 34 A. The distance of a phosphorus atom from the fibre axis is 10 A. As the phosphates are on the outside, cations have easy access to them.

Everything important about the structure of DNA is contained in this paragraph except for the base pairs. Note how important it was to confirm that the nucleotide conformation is similar to that which Furberg saw in the structure of cytidylate.

The important points about the backbone chains are that there are only two of them, that they form a regular helix, and the chains run in opposite directions. Recall that it was Crick who recognized the the chains had to be anti-parallel and nobody else, including Franklin and Wilkins, had thought of this.

The structure is an open one, and its water content is rather high. At lower water contents we would expect the bases to tilt so that the structure could become more compact.

This is an oblique reference to the A form of DNA that Rosalind Franklin was working on. The A form is somewhat dehydrated and the helix is more compact. Just as Watson & Crick predict, the bases are tilted in the A form.

The novel feature of the structure is the manner in which the two chains are held together by the purine and pyrimidine bases. The planes of the bases are perpendicular to the fibre axis. They are joined together in pairs, a single base from one chain being hydroden-bonded to a single base from the other chain, so that the two lie side by side with identical z-coordinates. One of the pair must be a purine and the other a pyrimidine for bonding to occur. The hydrogen bonds are made as follows: purine position 1 to pyrimidine position 1; purine position 6 to pyrimidine position.

If it is assumed that the bases only occur in the structure in the most plausible tautomeric forms (that is, with the keto rather than the enol configurations) it is found that only specific pairs of bases can bond together. These pairs are: adenine (purine) with thymine (pyrimidine), and guanine (purine) with cytosine (pyrimidine).

The pairing of A with T and G with C to form base pairs in the middle of the helix is the most important part of the proposed structure. It could not have been determined from the X-ray diffraction data. It could only be deduced by model building. Note that Watson & Crick emphasize the correct tautomeric forms of the bases since most of the textbooks of the day showed the incorrect forms.

In other words, if an adenine forms one member of a pair, on either chain, then on these assumptions the other member must be thymine; similarly for guanine and cytosine. The sequence of bases on a single chain does not appear to be restricted in any way. However, if only specific pairs of bases can be formed, it follows that if the sequence of bases on one chain is given, then the sequence on the other chain is automatically determined.

This is the idea of complementarity that was very much in the air among the insiders. It's an entirely theoretical idea but the fact that the structure conformed made it all that much more elegant a solution. The "beauty" of the structure derives in large part from the fact that it explains so much.

It has been found experimentally^3,4 that the ratio of the amounts of adenine to thymine, and the ratio of guanine to cytosine, are always very close to unity for deoxyribose nucleic acid.

This is a reference to the Chargaff ratios.

It is probably impossible to build this structure with a ribose sugar in place of the deoxyribose, as the extra oxygen atom would make too close a van der Waals contact.

An insight that proved to be correct. The Watson & Crick structure explains one more thing that none of the other structures could explain.

The previously published X-ray data^5,6 on deoxyribose nucleic acid are insufficient for a rigorous test of our structure. So far as we can tell, it is roughly compatible with the experimental data, but it must be regarded as unproved until it has been checked against more exact results. Some of these are given in the following communications. We were not aware of the details of the results presented there when we devised our structure, which rests mainly though not entirely on published experimental data and stereochemical arguments.

Watson & Crick know full well that their structure is compatible with published data from Astbury (ref. 5) and Wilkins & Randall (ref. 6). They also know that some of the key features of their model, such as base pairing, cannot be verified by X-ray crystallographic data from DNA fibers fibres.

They make reference to the accompanying papers by Franklin & Gosling and by Wilkins, Stokes, and Wilson ("following communications"). This was a standard way of referring to papers that were in press but Watson & Crick have been criticized for not mentioning the authors by name, especially Rosalind Franklin and Maurice Wilkins.

The last sentence has been widely interpreted as somewhat disingenuous. Of course they were aware of the results, including many of the details that had not been published (see below). A great deal of the structure of the backbones was informed by the results from Franklin's unpublished X-ray images of B-DNA. It would have been much better if Watson & Crick had stated here—as a personal communication—that they had received information from Wilkins and Franklin.

It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.

Another very famous sentence from the paper and another classic example of understatement. Watson & Crick follow up on this with another Nature paper that describes how DNA replication should work. The fact that an obvious mechanism of replicating DNA is apparent from looking at the structure is another example of its beauty and elegance. These were the sorts of thing that made the structure so appealing to those who were working on these problems. On the other hand, they meant nothing to most biologists, many of whom were not inclined to believe the Watson & Crick structure when it was first published. Remember that for most biologists this was the first time they were confronted with the idea that DNA was important. Watson & Crick had know for years that DNA was the secret of life but the rest of the world still thought DNA was unimportant.

Full details of the structure, including the conditions assumed in building it, together with a set of coordinates for the atoms, will be published elsewhere

The "details" were published in The Proceeding of the Royal Society in January, 1954.

We are much indebted to Dr. Jerry Donohue for constant advice and criticism, especially on interatomic distances. We have also been stimulated by a knowledge of the general nature of the unpublished experimental results and ideas of Dr. M. H. F. Wilkins, Dr. R. E. Franklin and their co-workers at King’s College, London. One of us (J. D. W.) has been aided by a fellowship from the National Foundation for Infantile Paralysis.

One of the myths that has grown up about the discovery of the double helix is that Watson & Crick never acknowledged Franklin and Wilkins. This myth is due, in part, to the fact that Wilkins and Franklin are not mentioned in the body of the paper where it would have been appropriate (see above). However, they are clearly mentioned in the acknowledgments even though the reference seems to contradict their earlier statement about being unaware of unpublished results.

Nobel Laureate: Charles Louis Alphonse Laveran

 
 
The Nobel Prize in Physiology or Medicine 1907.

"in recognition of his work on the role played by protozoa in causing diseases"

Charles Louis Alphonse Laveran (1845 - 1922) received the Nobel Prize in Physiology or Medicine for discovering that malaria was caused by a protozoan that infects red blood cells. There are many who believe that Laveran should have been recognized before Ronald Ross who received the Nobel Prize in 1902 [Nobel Laureate: Ronald Ross]. The Malaria Site has a very nice description of Laveran and his work [Charles Louis Alphonse Laveran].

The presentation speech was supposed to have been given by Professor C. Sundberg of the Royal Caroline Institute on December 10, 1907 but owing to the death of of King Oscar II two days earlier, the award ceremonies for 1907 were canceled. The text was published.

The Staff of Professors at the Caroline Institute have this year awarded the Nobel Prize for Medicine to Dr. Charles Louis Alphonse Laveran, for his work on the importance of the protozoa as pathogens.

The Staff has thus chosen to single him out not only as the founder of medical protozoology, a branch of medicine that has reached a striking level of development in recent years; but also as the man responsible for experiments and discoveries - followed up until recently - which ensure his continued pre-eminence in this field.

To appreciate properly the importance of Laveran's investigations into the protozoan causes of disease, one must remember the state of this branch of science at the time of Laveran's earliest work, i.e. about 1880. The body of knowledge relating to the causes of infectious diseases was making rapid progress at that time in the field of bacteriology. Pasteur's «Theory of Germs» had provided the key to the riddle of fermentation processes, and its relevance to infectious diseases had been grasped. So several pathogenic bacteria had been discovered by 1880: those of anthrax and relapsing fever; other germs, such as those causing tuberculosis, glanders, pneumonia, typhoid fever, diphtheria, tetanus, Asiatic cholera, traumatic fevers, etc. were discovered one after another during the years 1880-90. All these germs were found to belong to the last category of the plant kingdom, the bacteria.

As a result, it was natural to look for the cause of marsh fevers, like malaria, among micro-organisms of that sort. Indeed, several distinguished bacteriologists believed themselves to be on the trail of such a microbe. We recall the so-called malaria bacillus of Klebs and Tommasi-Crudeli, found in the ooze of the Pontine Marshes.

When Laveran, in 1879, began his research at the military hospital of Bône in Algeria, he only set himself the task of explaining the role of the particles of black pigment found in the blood of people suffering from malaria. After 1850, when these particles, called melanins, were discovered, methods had been discussed of determining whether they were only to be found in patients suffering from malaria, or were present in other diseases as well. Laveran first set about solving this problem, which was particularly important to the diagnosis of malaria. During his investigations, Laveran not only found the particles he had been looking for: he also found some entirely unknown bodies with certain characteristics which led him to suppose that parasites were involved. His initial investigations were carried out on fresh blood without using chemical reactions or any staining process. He was none the less successful, using this primitive method of examination, in distinguishing and describing most of the more important forms adopted by these new bodies, which varied so much in their appearance. In 1882, he moved the scene of his investigations for a while to the dangerous marshy regions of Italy. There he again found the same bodies in the blood of people suffering from marsh fever, and his hope of having found the malarial parasite became a certainty. Laveran published his first great work on these parasites, Traité des fièvres palustres, in 1884. In this, he draws on 480 examined cases of malaria.

This work is the foundation on which subsequent investigations of marsh fever are based. Laveran showed that the parasites, during their development in the red blood corpuscles, destroy them; and the red pigment in the corpuscles is changed into the melanin particles mentioned above. He described all the main forms of this polymorphic parasite, even those which have subsequently been found to be different developmental phases of the parasite. Continuing his work, Laveran concerned himself in the first place with the important problem of the existence of these parasites outside the patient's body. To this end he examined the water, soil, and air of the marshlands, hoping to find the parasite. His perseverance was unrewarded. We should not, however, fail to recognize the merit of this work, despite its negative outcome, since it has fundamentally aided subsequent research. As far as Laveran was concerned, these apparently fruitless investigations led him to the conclusions which he expresses in the book of 1884, and has also maintained on a number of occasions, such as the Congress of Hygiene at Budapest (1894): that the marsh-fever parasite must undergo one phase of its development in mosquitoes, and be inoculated into humans by their bites. Laveran based his conclusion not only on the negative experiments already mentioned, but also on an analogy with the mode of transmission of the Filaria worm, which, according to Manson, is mosquito-borne. When Laveran was recalled from Algeria to Paris, and so forced to interrupt his work on malaria, he had already clearly formulated the problems that had first to be solved in this field.

The new parasite discovered by Laveran was not a bacterium. Although it was impossible to classify accurately, certain resemblances to other micro-organisms put it in the same group as the protozoa. We know how difficult it is to demonstrate the presence of malarial parasites in blood which has not been treated beforehand with the stains now in general use, but still unknown at the time of Laveran's discoveries, which make these small parasites more readily visible; so one can appreciate at their true value the insight and keen eye of Laveran, who never allowed himself to be misled by the simultaneous successes of bacteriology, or discouraged by the opposition met with from several quarters, notably from workers studying marsh fever.

However, little by little Laveran's theories made headway, and it can be said that the year 1889 marks the date when his discovery finally achieved recognition.

When Laveran had to leave the marshlands, he saw himself deprived of materials indispensable if he were to continue working on the still unanswered questions, i.e. those dealing with the parasite's developmental cycle, and its existence away from the patient. He then tried to solve them by an indirect approach, by studying animal parasites, especially those of birds: these parasites had only recently been discovered and showed resemblances to the malarial parasites. The numerous observations Laveran made in the course of this research cannot be indicated here: they belong by rights to the specialist sphere of interest. Now, as always happens after a notable discovery, workers multiplied in the new field. Some of the many workers who were able to continue Laveran's work on the spot, in marshy areas, were destined to reach the goal before Laveran by the indirect approach which he had indicated. Thus, in 1897 the American Mac Callum elucidated the sexual reproduction of these parasites; and, in 1898, the impressive work of Ronald Ross, the Nobel Prize winner for 1902, brought the mosquito theory from the realm of hypothesis into that of established fact. One can imagine the interest with which Laveran must have received the preparations sent to him by Ross from India in May 1898, and the joy with which he confirmed that Ross was in fact dealing with malaria parasites in the mosquitoes he was investigating.

Laveran's discoveries concerning malaria had the additional effect of focussing direct and vigorous attention on the hypothesis that other infectious diseases could be brought about similarly by protozoa. In the tropics especially, but in other areas as well, diseases have been recognized for a long time among men and animals, which are similar to malaria in many respects, such as impoverishment of the blood, loss of strength, and associated fever, but which, unlike malaria, are not affected by the classical treatment, quinine, and are clearly shown by the absence of marsh-fever parasites not to belong to the same group as the marsh sicknesses. Since 1890 a whole series of parasites causing these diseases has been described. Once, thanks to Laveran, attention was drawn to the protozoa as agents of disease, discoveries of such protozoa took place in rapid succession. Among diseases due to protozoa, the trypanosomiases take precedence. The list of these diseases alone is long, and we will mention only the scourges known as Nagana, Surra, Caderas sickness, and the Galziekte of Equatorial Africa, etc. which ravage large parts of Africa, Asia and South America, attacking various members of the Bovidae, horses, camels, donkeys, etc. as well as the big game, antelopes, deer, etc. sometimes wiping out great herds. All these infections are caused by corkscrew-shaped micro-parasites, called trypanosomes, and are transmitted to animals by various types of biting flies. However important these diseases may be to Man from the point of view of commerce and nutrition, yet, among all the trypanosomiases, the endemic disease generally known as «sleeping-sickness» takes precedence from the medical point of view. The sleeping-sickness trypanosome was discovered in 1901 by Forde in a European ship's captain who had navigated the river Gambia for several years. Forde does not seem to have examined the parasite in detail. Later, the same case was studied by Dutton, and following on his reports on the parasite and the disease, an expedition was sent from Liverpool and London to carry the investigation further. This expedition also solved the first problems relating to the disease. There is certainly much one could say about these diseases; unfortunately we may not dwell on them here. Let us rather take a quick look at the part played by Laveran in the elucidation of these problems.

It can be said, it seems to us, that Laveran took up these problems again at the exact point where circumstances had forced him to interrupt his own research on malaria. He had discovered the parasites for the latter group of diseases, but others, notably Golgi and Ross, followed up the biological investigation of the parasites. As far as the trypanosomiases are concerned, the opposite holds good: the parasites were discovered by other investigators, who were able to study the investigations on the spot in a number of different places, but Laveran, more than anyone else, extended our understanding of the finer points of the morphology, biology, and pathological activity of the parasites. He made this work possible by having many artificially-infected experimental animals brought to his Paris laboratory, as well as larger animals which had contracted the disease naturally. Not content with this great quantity of material, he extended the scope of his investigations even further by studying the trypanosomes of rats, birds, fishes and reptiles; and these investigations often threw light on the true pathogenic trypanosomes at the same time. The trypanosomes thus studied and described by Laveran number about thirty; he discovered a greater number of new species than any other worker we know of. In addition, he discovered a new genus of trypanosomes, the trypanoplasmias.

Laveran published his discoveries, sometimes in collaboration with other workers, in many articles and annotations, and later, in 1904, he gathered them together in one great work, so far unique of its kind: Les trypanosomes et trypanosomiasis.

Still more recently, in 1906, there appeared the accounts of his research on the parasites causing the malignant Mbori, Souma, and Baléri diseases, which are widespread among the Bovidae, camels and horses of the Upper Niger.

It is obviously impossible to compress into a few words the rich content of all his writings, his investigations, and his numerous discoveries. In them we find technical inventions for the study of parasites, morphology, theories of infection, accounts of parasite reproduction, experiments in immunization, etc. These works are proof that the creator of protozoan pathology continues to be its leading authority. For these reasons and many others that could be added, the Staff of Professors of the Caroline Institute have pleasure in awarding this year's Nobel Prize to this pioneer of science, this tireless benefactor of humanity.

Nobel Laureate: Ronald Ross

 
 
The Nobel Prize in Physiology or Medicine 1902.

"for his work on malaria, by which he has shown how it enters the organism and thereby has laid the foundation for successful research on this disease and methods of combating it"

Ronald Ross (1857 - 1932) received the Nobel Prize in Physiology or Medicine for discovering that the malaria parasite was transmitted by mosquitoes. You can read a detailed description of Ross' work on The Malaria Site [Sir Ronald Ross]. He was a remarkable man.

This Nobel Prize—only the second one to be awarded—was controversial. Read the presentation speech by Professor the Count K.A.H. Mörner of the Royal Caroline Institute below and note the mention of several other workers, including Patrick Manson, Ross' mentor, and Alphonse Laveran, who discovered the malaria parasite. There are many who believe that Manson and Laveran should have received part of the prize. Laveran was recognized five years later when he received his own Nobel Prize [Nobel Laureate: Laveran].

Your Majesty, Your Royal Highnesses, Ladies and Gentlemen.

Among the stipulations Alfred Nobel set forth in his will, on which the Nobel Foundation is based, that concerning the international character of the prizes occupies an important place. This proves not only his love of mankind and his wish that we should regard one another as brethren, but it is also a witness of his extensive and prescient views more especially concerning medical science and its advancement.

All the branches of medical science and their promotors in different countries have the same ultimate aim, that of gaining the most thorough knowledge possible both about the human body and the processes in it, as also about noxious influences and the means of their prevention. All medical workers unite in pursuing that aim and in doing so feel members of one great fellowship. Nevertheless, the different fields of medical science lie at such a distance one from another, that the individual worker on many occasions must look afar in the attempt to get a thorough view of the progress of the work.

With respect to diseases they are often of different kinds and import in divers regions of the world. For instance, malaria is nowadays of little importance here in Sweden, whereas it is a veritable scourge in other regions. For elucidating this question by an instance from a European country, it may be mentioned that in Italy of late the annual average of deaths by malaria has been about 15,000, and the yearly number of cases is calculated as about two millions. Still more overwhelming are the numbers from India. Of the British Army, amounting to about 178,000 men, close upon 76,000 men were admitted into hospital for malarial fever in the year 1897. In this single year the mortality from «fever» among the civil population in India amounted to a total of more than five millions. It is moreover a well-known fact, that malaria dominates so severely in vast territories that it causes the very greatest difficulties for the cultivation of countries which, but for the malaria, are specially favoured by Nature.

The question of the real nature of malaria, its origin, its manner of entering the organism, and the consequent question of the possibility of preventing this disease, are all of the greatest importance and have from remote ages occupied investigators, for a long time without success.

A very important discovery concerning malaria was made - now long ago, more than two decades - when Laveran, a French army surgeon, ascertained, that malaria is a parasitic disease, caused by a very low form of animal life, that he found in the blood of malarious patients. By this discovery the name of Laveran has for ever become renowned in the history of malaria.

Research about malaria in the last two decades has chiefly been based on Laveran's discovery. Science has thereby been enriched with many an important fact. We have gained knowledge of the different forms of the malarial parasite in blood. We have found, that it differs in the special forms of the disease. We have learned the relations between the parasite and the red blood corpuscles, in which it is chiefly to be found. We have furthermore been able to survey the manner in which it multiplies in the blood; the Italian investigator Golgi has in this respect revealed the remarkable fact that the periodicity of the malarial attacks depends on the appearance of new generations of the parasite in the blood. We have moreover found allied parasites in the blood of several mammals and birds.

The important question, previously mentioned, as to the possibility of the malarial parasite living outside the body, and its way of obtaining entrance into the blood remained unanswered. For some reasons, among others owing to various facts that were known concerning other parasites of an animal nature, it was supposed that the malarial parasite in some way leaves the blood so as to exist in some form in nature, probably as a parasite of some other being. As nothing indicated that the parasite was to be found in the secretions or excretions, the supposition lay near at hand, that suctorial insects would assist in carrying the parasite to a place, where it had to pass the aforementioned part of its life-cycle. Attention was therefore directed to the mosquito, which was thus supposed to spread the malarious infection. The importance of the mosquito in this respect has now been proved. In this case, as in several others, tradition anticipated science; it is even said, that negroes in the East-Africa use the same name for the mosquito and for malaria.

The mosquito theory of malaria was introduced to science by King no less than 18 years ago. The theory, however, remained a conjecture without other evidence than some suggestions given by epidemiological observations. The attempts made in Italy in the early nineties with the view of examining the theory experimentally, and, eventually, proving it to be true, gave results that seemed anything but encouraging; being far more likely to prevent the investigators from following this line.

A person we deem of great merit concerning the solution of the problem is the English investigator, Patrick Manson. It was a change in the appearance of the parasite, which was sometimes observed to occur, as the blood is shed, that Manson especially regarded as the first stage of its life outside the body. This phenomenon has afterwards been shown by the American pathologist Mac Callum to imply an act of reproduction of the parasite. Manson was moreover guided by his experience regarding another parasite of the blood, a little worm, filaria, the transference of which from one part of its life-cycle to another he had found effected by the mosquito, and more particularly by special species of the mosquito. By his views set forth on malaria, and by exciting expectation that the solution of the malaria problem was to be found in the direction he indicated, Manson gave an impulse to the further testing of the mosquito-theory and at last to its being established. Manson, who lived in England, had no opportunity of taking up the experimental work of the problem. The solution came from India.

It was an English army surgeon in India, Ronald Ross, who, impressed by Manson's induction, undertook the experimental testing of the matter. Critically arranging his experiments, he caused mosquitoes that were hatched from larvae in the laboratory, to bite malarious patients, and endeavoured to follow the parasite in the body of the mosquitoes. The results of the first two years' labour, although assiduous and scrupulous, gave little promise of success. But in August 1897 all at once he made vast progress towards his aim. While experimenting with another, less common species of mosquito, in the wall of its stomach he found bodies that very probably were an evolutionary stage of the human malarial parasite.

Ross, being prevented by circumstances from pursuing his plan in studying the malarial parasite of man, continued his work with an allied malarial parasite of birds. The result was that not only could he confirm his discovery concerning human malaria, as he found corresponding facts for avian malaria, but he also in a short time succeeded in revealing the further development of the avian malarial parasite in the body of the mosquito.

This development is briefly as follows. In the stomach of the mosquito a process of fecundation at first takes place; the form of the parasite, thereby produced, penetrates the stomach wall, embedded in which it grows to button-like structures projecting into the body-cavity. In these structures a large number of elongated organisms, «sporozoites», are formed. On the consequent bursting of the said structures the «sporozoites» escape into the general body-cavity of the mosquito, and accumulate in the salivary or poison glands, which are in connection with the proboscis with which the bites of the insect are inflicted. A bite of the mosquito, at that time, inoculates the parasite, and if the individual is susceptible to the parasite, this develops in the manner known and described long ago.

Ross's discoveries into malaria were immediately followed by a series of important works.

Thus the Italian investigator, Grassi, in association with his colleagues, Bignami and Bastianelli, proved that the human malarial parasite not only in its early stage, already detected by Ross, but also in its further development undergoes the same evolution that Ross described for the growth of the avian malarial parasite in the body of the mosquito. Grassi also has precisely indicated the species of mosquito that are of import for the malaria of man. Many valuable works, besides these, have been issued by Ross, by the Italian investigators, by Robert Koch and by many others, works, by which not only our knowledge of the malarial parasite has been enlarged, but this knowledge has been made useful in combating and preventing malarial disease.

The eminent scientific value of Ross's work, its importance as a basis for the success of the recent investigations into malaria, its rich contents as regards the art of medical practice and especially hygiene, will be obvious from the above.

It is owing to these merits, that the Professorial Staff of the Royal Caroline Institute has decided to allot the Medical Nobel Prize of this year to Ronald Ross.

Professor Ronald Ross. In announcing that the Professorial Staff of the Royal Caroline Institute has decided to award to you the Medical Nobel Prize of this year on account of your work on malaria, in the name of the said Institute I congratulate you on your investigations. By your discoveries you have revealed the mysteries of malaria. You have enriched science with facts of great biological interest and of the very greatest medical importance. You have founded the work of preventing malaria, this veritable scourge of many countries.

Plasmodium falciparum Causes Malaria

 
Malaria is caused by a small protozoan parasite called Plasmodium falciparum (left). The Plasmodium is a single-celled organism with a complex life cycle. It is classified in the nebulous Protist kingdom within the phylum Apicomplexa [NCBI Taxonomy].

The life cycle is described in many places but one of the best comes from the Applied Biosystems website.

Human malaria is caused by infection with intracellular protozoan parasites of the genus Plasmodium that are transmitted by Anopheles mosquitoes. Four species of Plasmodium infect humans: P. falciparum, P. vivax, P. ovale, and P. malariae, with P. falciparum accounting for the majority of infections and being the most lethal. The causative agent of malaria was discovered in 1880 by Charles Alphonse Louis Laveran (Ref.1).

Plasmodium falciparum is exclusively transmitted by female Anopheles mosquitoes, mainly from members of the Anopheles gambiae complex. The parasites have a complicated life cycle that requires a vertebrate host for the asexual cycle and a female Anopheles mosquito for completion of the sexual cycle. Infection of humans by P. falciparum is initiated by injection of sporozoites into the bloodstream by an Anopheles mosquito (Ref.2). During a mosquito blood meal,infectious Sporozoites in the mosquito's saliva enter the host bloodstream and invade its hepatocytes. While some evidence indicates that Sporozoites are first trapped by Kupffer cells and then transported to hepatocytes,other findings suggest that Sporozoites home to hepatocytes directly. Sporozoite reaches liver via bloodstream in 30 minutes....

In the hepatocytes asexual multiplication (exoerythrocytic schizogony) occurs, leading to the production of several thousand merozoites. In 1 to 2 weeks, a single sporozoite can give rise to 30,000 merozoites. During this pre-erythrocytic stage,no illness is induced by malaria. In P. vivax infections, which are characterized by relapses,a dormant stage, called the hypnozoite, remains in the liver. From this stage relapsing infections may occur at a later stage. P. falciparum infection relapses do not occur. It is, therefore, assumed that the sporozoites of this species develop uniformly producing pre-erythocytic schizonts at the same time and these schizonts, once formed,discharge all the merozoites simultaneously; do not remain dormant as in P. vivax (Ref.3).


These Merozoites are released into the bloodstream and invade erythrocytes. The asexual erythrocytic cycle begins when a single merozoite invades a host red blood cell and is enclosed within a parasitophorous vacuole,separate from the host cell cytoplasm. Three morphologically distinct phases are then observed. The ring stage,lasting approximately 24 h in P. falciparum, accounts for about half of the intraerythrocytic cycle, but it is metabolically nondescript. It is followed by the trophozoite stage; a very active period during which most of the red blood cells cytoplasm is consumed. Finally,parasites undergo 4-5 rounds of binary divisions during the schizont stage, producing 8-36 new merozoites that burst from the host cell to invade new erythrocytes,beginning another round of infection. This phase of the infection (erythrocytic schizogony) is responsible for malaria pathogenesis. Much of the morbidity and mortality associated with malaria is caused by the rupture of iRBCs (Infected Red Blood Cells) during the asexual reproductive stages of the parasite. Intense fever, occurring in 24-72 hour intervals, is accompanied by nausea, headaches,and muscular pain among other symptoms. The characteristic fever spike has been correlated with incremental rises in serum levels of TNF-Alpha associated with the release of parasite proteins during erythrocytic rupture. Furthermore,a variety of potentially fatal symptoms,including liver failure, renal failure,and cerebral disease are associated with untreated P. falciparum. These symptoms are consequences of the unique ability of the parasite to bind to endothelial surfaces; this adherence inhibits circulation and causes localized oxygen-deprivation and sometimes hemorrhaging. It has been proposed that ICAM1 (Intercellular Adhesion Molecule-1), E-selectin,VCAM1 (Vascular Cell Adhesion Molecule-1), and CSA (Chondroitin Sulfate-A), and CD36 are some of the surface molecules responsible for parasite-endothelial adherence (Ref.4).

Instead of producing new schizonts, some merozoites, after invasion of the erythrocyte, arrest their cell cycle and develop into male (micro) or female (macro) gametocytes, the forms that are required for transmission to the mosquito (asexual parasites do not survive ingestion by the insect). Inside the mid-gut of the mosquito, fertilization occurs, producing zygotes, which develop into ookinetes. The ookinetes form oocysts, which then grow and divide and rupture to give rise to sporozoites, which migrate to the salivary glands. Then the infectious cycle of malaria can repeat itself (Ref.5). While all four species of Plasmodium have a haemolytic component ie. when a new brood of parasites break out of the red blood cell this is usually of little consequence. The exception is falciparum malaria where the parasites multiply very rapidly and may occupy 30% or more of the red blood cells causing a very significant level of haemolysis. One reason for this is that P. falciparum invades red cells of all ages whereas P. vivax and P. ovale prefer younger red cells, while P. malariae seeks mature red cells. Malaria places an increasing burden on global public health resources. In the face of growing resistance of the malaria parasite to available antimalarial drugs, there is a need for new drugs and the identification of new chemotherapeutic targets (Ref.6).

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Image Credits:

Plasmodium falciparum, the parasite that causes malaria in humans, needs a living host in …. [Photograph]. Retrieved July 25, 2007, from Encyclopædia Britannica Online: http://www.britannica.com/ebc/art-55545

Life Cycle diagram is from Don Forsdyke.

The red blood cell image is from The Scripps Research Institute.

Quinine and Malaria

 
Monday's Molecule #36 was quinine, an alkaloid isolated from the bark of Chichona, or quinine tree [Cinchona pubescens]. The tree originally grew only on the eastern slopes of the Andes in South America where the bark was widely used by the natives to prevent malaria and other diseases. Following the discovery of its amazing properties by Europeans, it was transported to other tropical parts of the world.

Quinine works by attacking the parasite that causes malaria. This protozoan parasite, Plasmodium falciparum, feeds on red blood cells. It can easily digest hemoglobin but can't handle the heme groups that are released when the protein is degraded. These heme groups are toxic to the parasite so they are stored in an inactive form inside a membrane-bound organelle called a digestive vacuole. Quinine interferes with this storage causing the hemes to remain free where they poison the cell. The exact mechanism is unknown but it is known that quinine has to enter the vacuole in order to be effective. The most likely mechanism is quinine binding to the heme molecule to prevent its conversion to the inactive form celled haemozoin.

Resistance to quinine and related compounds is usually due to mutations in transporter proteins that are found in the membranes of the digestive vacuole. The mutations prevent the accumulation of quinine in the vacuole.

Quinine is present in tonic water that was widely consumed in the last century to ward off malaria. The quinine imparts a bitter taste to tonic water so, as the story goes, British tourists used to dilute it with gin to hide the taste. The gin & tonic mixture became quire popular.

As a matter of fact, quinine is still present in modern bottles of tonic water. This can be easily demonstrated by shining ultraviolet light on a bottle of tonic water since quinine is fluorescent (left). To see how much quinine you get in a gin & tonic see [The Half-Decent Phamaceutical Chemistry Blog].

Quinine was synthesized after World War II but it isn't economical to make the drug and the only effective source is the bark of Chichona. However, a more effective drug called chloroquine (below) became widely available after World War II and it has mostly replaced quinine as the preferred drug against malaria.