Professor Hoppe and friends, I find professors Hoppe's introduction very useful to me,
I should be illustrating some of the remarks he has made in the course of this lecture.
As you have heard already from other speakers, discoveries often get lost in the literature,
well, perhaps Dickinson really showed you how to find them, however far back you had to go.
And sometimes observations that should lead on to great developments get made and somehow not used,
there are gaps of ten, fifteen, twenty years, before they are really, finally put to good use,
that everybody works on the problems revealed, as in the case of interferon yesterday.
And these gaps are, in themselves, quite interesting, and they occurred in the course of the story of the x-ray analysis of proteins.
Protein crystals were observed in plants and in animal tissues during the course of the 19th century,
and there are many nice drawings of them made by botanists and others who examined biological tissues.
The first one, on my first slide, was made by Professor Schimper
and I think from this part of Germany, pictures of crystals observed in plant cells,
and you can see, he was looking at them through microscopes and he, there is one,
particularly here - is there a pointer? -
which you can see, he has viewed through nickels in different directions, which shows pleochroism.
And some of them are protein crystals.
I'm meaning this one here where he's obviously got his nickels in different directions.
And the, another one on the next slide, also taken from more than a hundred years ago, from Preyer's book on "Die Blutkristalle",
a very lovely photograph of haemoglobin crystals, I think they are dog haemoglobin,
showing that he was viewing them through the microscope using nickels, turning them round
so that the crystals appeared different coloured in different directions.
And even at that time it was realized that proteins, that molecules, whatever they were, in these crystals were large.
On the next slide there is a little early analysis made, figured in Preyer's book,
giving a shot at the molecular weight of haemoglobin.
It isn't quite right in any direction because the iron analysis is too high, and, but it gives you see a large figure of 13.000,
it should be more like 17.000, and then the actual molecule is four times that.
But it was known that there were large molecules in the crystals in the 19th century,
and observations were made by both Schimper and Preyer, that showed that to get these beautiful pictures of crystals,
you must keep the crystals covered with liquid.
And that they dried or shrank when removed from their mother liquor.
Now the next discovery was the discovery made in Munich by Von Laue, Friedrich and Knipping, illustrated by the next slide.
Oh sorry, this is just another one that shows you a crystal actually growing,
a crystal of haemoglobin actually growing in a red blood cell,
and you can see that the haemoglobin crystal is occupying almost the whole of one of the blood cells.
It's this particular cell, probably the wall was damaged, and so the crystal started to grow,
whereas in the next one you can see the normal appearance.
And now the photograph which was taken by Von Laue, Friedrich and Knipping in 1912 in Munich
by passing x-rays through copper sulphate,
and it shows that x-rays have wave lengths of the order of magnitude of diffracting units in the crystal,
and that these units must be atoms arranged in a regular arrangement in three dimensions to produce these effects.
Von Laue, Friedrich and Knipping didn't go on to work on this crystal, its structure wasn't solved for more than twenty years.
It seemed quite complicated in those days, they moved over to a cubic crystal, zinc sulphide, and took very beautiful photographs.
But the actually first use made of these x-ray diffraction photographs was made by a very young man,
W.L. Bragg, age twenty four, in England, who showed how to use the diffraction effects
to find the relative positions of the atoms in space in sodium chloride.
He was helped by the structure having been suggested to him by Barlow,
who in fact had published a proposed structure in 1885, quite correctly, again a long time before.
I illustrate the structure on the next slide by an actual section in the electron density in the crystals of sodium chloride.
The electrons scatter the x-rays and because they are grouped into atoms, in a regular arranged three dimensions,
the interference is partly destructive, and from the spectra one can form a Fourier series as W.H. Bragg first suggested in 1915.
The spectra provide the components, the terms of the Fourier series, they, from the scattering separates the terms,
they have to be recombined to give you back the pattern which produced them.
And the recombination has to be done, is usually done mathematically
by calculating the contribution of every term observed to every position in the crystal by a mathematical formula,
and for this you have to know the amplitudes of the waves, which you can easily measure, and also their relative phases,
which are lost in the process but can sometimes be easily recovered.
The calculation, though suggested in 1915, was not in fact made till 1926 by Habbicurst in America,
and Dewan who suggested it, pointed out that the phases were, one, known in sodium chloride from Bragg's work,
but two, could have been inferred because the heavier atom would dominate the effects,
or alternatively, as Bragg had used to begin with, the differences between sodium chloride and potassium chloride,
where one ion varied in density, would give a direct method of finding the phase relations.
And then the picture can be combined and the electrons plotted,
the electron density plotted at any density intervals you liked, to show the arrangement of the atoms.
Now, when the experiments on x-ray diffraction were first made, passing x-rays through crystals,
it was natural for different people in different parts of the world to repeat the experiments, W.L. Bragg was one.
But they were also repeated in Japan, and in Japan, for the first time in 1913, immediately afterwards,
x-rays were put through a protein, silk fibres.
And I think the next slide should show you a photograph of silk fibroin.
Now, this is a photograph in which the reflections are very fuzzy.
Good photographs were obtained first actually in Berlin in 1921/22 by again a very young man, as he was then, Rudolf Brill,
who I hope is still alive and living near Munich, as he was a year or two ago, and he took the photographs for his dissertation,
helped in the interpretation by Michael Polanyi and Hermann Mark, who were slightly older, in the same laboratory.
And the interpretation was that in these fibres there must be long chains of proteins, as indicated by Emil Fischer's experiments,
and that these chains were not quite regular, that the amino acids might not repeat quite regularly.
So this isn't a perfect crystalline photograph, but one in which the essential intervals
shown by these fuzzy spots, are the intervals in the chains.
And the next slide shows Professor Mark and Meyer's idea of what the protein chains, amino acid chains should be like
to give the actual observed distances between these fuzzy reflections on the silk fibroin photographs.
Extended zig-zag chains alternately glycine and alanine in the fibre structure, running through the unit cells.
In this period of the 1920s, there were a number of experiments in which crystals were actually prepared in the laboratory
from newly isolated enzymes and hormones, urease, Sumner, Northrop, pepsin, insulin by J.J. Abel in America,
and it was natural for young crystallographers in the 1920s to try to put x-rays through these crystals too.
So in the laboratory of W.H. Bragg at the Royal Institution,
several attempts were made to get x-ray photographs of insulin, haemoglobin, one of the enzymes, edistin, a plant hormone,
and they all got nothing but somewhat vague blurs.
Two of the young men present were Asbury and J.D. Bernal, and when they left the laboratory of the Royal institution,
Bernal for Cambridge and Asbury for Leeds, to work on wool fibres at York, they were both very anxious to work on proteins.
And they corresponded with one another, and their correspondence exists in the Cambridge University Library where I found it.
And Asbury described how he wrote to Northrop for pepsin crystals and Northrop sent him ones
and he got absolutely nothing on the photographs, except a sort of, he got two rather diffuse reflections, rather like some of the silk fibroin ones.
And he took fibre photographs as well;
in fact that particular silk fibroin photograph is taken by Asbury and not by Brill or anyone of the earlier workers.
He found that protein fibres in general tended to give two patterns, one hair when it was un-stretched with two reflections
which he called the alpha pattern, and then if you pulled it out, it gave the pattern that suggested stretched chains, the beta pattern.
But he was really anxious to work on crystals and to collaborate with Bernal,
the only thing he complained of in his letters was he would like to start a serious collaboration,
if only you were not such a soft-hearted chap, and taking on problems for all sorts of other people.
And the problem of course that J.D. Bernal was taking on at that particular moment was the structure of the sterols.
He had just put x-ray photographs, x-rays through calciferol crystals and shown from his results
that the Wieland-Windaus formula couldn't be correct, and so opened the way for a whole new passage in sterol chemistry.
But Asbury wrote, why not ask for haemoglobin crystals, a dare is the bloke, and Bernal, I think, hesitated a little,
but suddenly the crystals were brought to him in his hand.
They were brought from Uppsala, where they had been grown by a young man called John Philpot,
who was a biochemist, learning how to purify proteins with Tiselius.
And John Philpot enjoyed skiing.
He went off skiing in the mountains for a fortnight, leaving his crystals growing in the fridge, and when he came back,
he found his tubes of purified pepsin full of the most marvellous large crystals, about two millimetres long.
And as good fortune for the advance of science would have it, they are passed through the laboratory, Glen Millikan,
the son of R.A. Electron Millikan, who was working in Cambridge on fast reactions,
and he was shown the crystals and he said, I know a man in Cambridge who would give his eyes for those crystals.
And Philpot happened to know the same man, John Desmond Bernal,
because he had earlier been involved in the isolation of vitamin D at the medical research institute in our country,
and so he, very willingly, handed him a tube of the crystals, which Millikan stuck in his coat pocket right way up,
crystals still in their mother liquor, and took them back to Cambridge.
The year was then 1933, and when Bernal saw the crystals, of course he immediately did,
he looked at them first within the tube under the microscope, and saw they were brightly shining, brightly birefringent,
and he took one out of it, being in a hurry to see what was happening, just with a needle out of the tube,
and took an x-ray photograph of it, and got exactly what Asbury had got, only perhaps rather less
because he was a less skilful in general experimenter, hardly anything on the photograph.
And he thought, this must be wrong, went back and looked at the crystals, bright in their mother liquor,
and it suddenly struck him that they needed their mother liquor round them to keep their actual form.
And he was lucky in another way because he was working at that time also on the problem of ice and water,
and he had in the laboratory a student, Helen Megaw, taking x-ray photographs of ice crystals
which she grew in little fine Lindemann glass tubes and kept at low temperatures.
So Bernal took just one of her little fine walled tubes, about half a millimetre across,
and fished out a pepsin crystal within its mother liquor, sealed the tube at two ends and put x-rays through it
and immediately got an x-ray photograph with reflections, ever so many reflections all over the photograph.
Now, the next slide should, if I am remembering right, well, first we have the people,
so here is J.D. Bernal much later on in life, and talking to Kathi Dornberger,
a student who was working at that time with V. M. Goldschmidt in Göttingen,
but came to work with him and came to work on some of the protein problems later, and myself,
and I must say, this photograph was taken in relatively old age,
when Kathi had just become the director of a small institute for x-ray diffraction studies in Berlin East.
Now, the next slide shows another character in the story whom I will mention, A.L. Patterson, in his laboratory with two students.
And the next slide shows the photographs, this isn't the original pepsin photograph,
here are some pepsin crystals swimming about in their mother liquor,
and above is a photograph taken of them by Professor Tom Blundell in Birkbeck College, London,
showing very, very many reflections along these parallel lines on an x-ray photograph.
The original photographs we think must have perished at Birkbeck since the laboratory,
part of the laboratory was destroyed during bombing during the war,
but it, this at least illustrates the character of the picture, quite different from silk fibroin, a definite crystal repeat,
you can very easily measure one that is constant, 67 angstroms from the separation of the lines,
and the other one, in fact, we got it wrong when first we measured it, we got it about half the size it really is,
it's really nearly 300 angstroms, corresponding to the long dimension of the pepsin crystals.
I was at that time working with Bernal, and it was only a sort of bit of bad luck, but perhaps it was good luck for science,
that I was not in the laboratory the day the first crystals came in, I was having a bad cold or something,
and Bernal made all of the first observations himself.
I'm always a little afraid I might have got more on the first photograph
since it was possible to get more reflections from the dried crystals than Bernal actually did,
and so delayed the observation that it was absolutely necessary to keep these crystals in their mother liquor.
But I went on to take most of the rest of the photographs, but I do some of the calculations
which we didn't carry very far because our first measurements indicated that we had a very large unit cell,
that it could correspond to there being 12 pepsin molecules in this cell, each of weight about 40.000,
several thousand atoms each you see within each molecule, and that this was,
it was beyond our possible means at that time to think that we could work out the structures of such molecules.
And the, yet the reflections extended to about 1 1/2 angstroms, it was clear that they were sufficient to show us atoms
if ever we could form an electron-density pattern from them and look at it.
At that time I was under pressure to return to Oxford to a college teaching appointment that should lead to a permanent appointment.
I was most unwilling to go but everyone in Cambridge said, difficult to get university jobs in this time,
of course you must take it, so reluctantly I went back to Oxford.
Bernal had got a small grant to support me, of 200 a year, which he gave to another young person and I think
- could I have the next slide? - and this was Isidore Fankuchen, who then was visiting from America,
who had come over with W.L. Bragg and was working with W.L. Bragg at Manchester,
and heard about the work at Cambridge and wanted to join in.
And he came in to take the next protein crystal, Chymotrypsin from Northrop, and then passed over to work on virus crystals
that played a very important part in the development of the subject, and particularly later in America.
And the next year there came another young person to work with Bernal in Cambridge and I think he's shown the next slide,
again much older than he was, Max Perutz, and Max Perutz came from Vienna wanting to work with Hopkins,
but Mark had forgotten to ask Hopkins to have Max Perutz as a research student when he visited Cambridge in 1935,
because he was so excited by the work that Bernal was doing and sent him instead to work with Bernal,
saying there's someone who really needs you, and Max said, but I don't know any crystallography,
and Mark said, you will learn, my boy, which he did, in the hard way, for many years to come.
The other one in the picture is John Kendrew, who Professor Hoppe mentioned,
and he doesn't come into the story for very much longer.
Now, what happened to me in Oxford, working, going back to begin work all on my own, was that Sir Robert Robinson
who was then Professor of organic chemistry, was given a small present of the first insulin crystals
obtained by the firm brutes in our country following a prescription for growing insulin crystals given by D.A. Scott in America,
that it was necessary to add zinc to the preparation.
They gave Robinson 10 milligrams in a little tube, and he hadn't any use for them, and knew the work that we had done in Cambridge
taking x-ray photographs of pepsin crystals, so he said, why don't you try to photograph these?
And they were microcrystalline but very bright and birefringent, and so I looked up all the preparations
and grew the crystals finally, not very well, by Scott's method, large enough to take x-ray photographs of,
and I made a horrible mistake, I decided that it didn't matter whether they were wet or dry and it was easier to handle them dry.
I dried them like good organic chemists did, pouring methyl alcohol over them and then took x-ray photographs of them.
And these very dry looking crystals, as you can see, are the crystals, not looking very good single crystals but they are,
and up at the top is the little x-ray photograph they gave.
Well, they gave an x-ray photograph, spots on the film, and I developed the first x-ray photograph about ten o' clock at night,
and waited in the lab while I fixed it and washed it, and then walked out absolutely dazed, very excited, little spots on this photograph,
down through the centre of Oxford, away from my lodgings
and about midnight I was accosted by a policeman who said: "Where are you going?"
So I said, not very truthfully: "Back to college", and turned round and went back.
But I woke up in the morning, next morning, about six, and I was suddenly extremely worried, and I went to the -
I thought, perhaps those spots, perhaps those crystals aren't really protein crystals at all, but something else,
some impurity, some breakdown product in the preparation.
And I went down very quickly before breakfast to the laboratory and picked one out of the tube and tried protein tests on it,
and I tried the xanthoproteic reaction, which consisted of dropping first a drop of concentrated nitric acid and it turns yellow,
and then a drop of ammonia and it turns brown, which it did, to my great relief,
and I went back, happily, to breakfast.
Now I perhaps should tell all those who are young here:
Why I knew that reaction so well was because I was rather young and still at school
I had a laboratory and did experiments on my own,
and I was doing experiments suggested by Parson's Fundamentals of Biochemistry,
and completing something one Sunday in a nice new silk frock, and of course one should never do this kind of thing,
and I accidently dropped a spot of nitric acid on the front of my dress, and seized the nearest alkali
which was ammonia and put it on it, well, of course it was much worse, and I was dreadfully upset,
my mother comforted me and said she could cover it all with a frill, which she did,
and so this particular reaction is indelibly engraved in my mind
and I was very pleased when I could test it once again with the insulin crystals.
Now, what happened after that?
I didn't really remember until I was back reading the Bernal files at Cambridge, but it's obvious that directly after breakfast,
I rang up the Cambridge Lab to tell them that I had taken these insulin photographs
and got the very sad news that Bernal was at home with a temperature of 104.
So then I wrote a little letter to his wife saying, please tell him when he's well enough that I have these insulin photographs,
and I gave the rough dimensions of the crystal unit cell, and they are on the next slide.
A rhomb is the real form of the crystals, 74.8 across 30.9 high, and within the crystals,
there's roughly 36.000 molecular weight of protein, and it should formally be divided into three,
which by the crystal symmetry to give you 12.000 molecular weight for the insulin molecules in the unit cell.
Bernal recovered and wrote me a letter which begins "Dear Dorothy, ZN 0.52%, CO 0.49%, CD 0.74%.
This gives rather less than three in each case.
I am going back to Cambridge on, I forget when, I will send you some cadmium stuff"
and any crystallographer can see what he was saying in this letter.
This zinc, according to D.A. Scott's observations, is replaceable by other elements,
of which cadmium is the heaviest so far observed, you should try
and see if the cadmium crystals show changes in the intensity of the x-ray reflections
and then you might be able to use the method of isomorphous replacement
to determine phase constants for the different reflections and really see the atoms in your crystal.
Terribly premature, I'm afraid, again I didn't remember all the details, I found the letter in which I said,
"sorry I can't get anything like good crystals from the cadmium material, it must be very impure,
I'm having a terrible time with scholarship examining for the college", and I did make one or two abortive efforts.
But I had a feeling that cadmium wasn't really heavy enough to do what was wanted
and that anyway, if I did a little calculation on the number of atoms, that there were in insulin.
It was too large a problem for myself to set out to work on at the age of 24, and that I must try to solve some simpler structure first.
I tried out the idea of the isomorphous replacement,
again actually in little calculations at the Royal Institution in a notebook on cholesterol, chloride and bromide,
while I was taking the insulin photograph which is shown on the previous slide,
because it's a very, I took that particular photograph for show, for publication in the Royal Society,
using the very big x-ray tube at the Royal Institution for the purpose.
So, I didn't go on with insulin, but Max Perutz went on with haemoglobin.
Could I have the next slide please?
I went on, as I said, with sterols, and with the sterols I explored the possible use of both
heavy atoms and isomorphous replacement for showing electron densities, and this is one of the experiments we did.
And I show it because I have to introduce another character in the story, and this is A.L. Patterson.
A.L. Patterson, I showed on that earlier slide, was one of the young men, A.L. Patterson, Asbury and Bernal ...