Teachers Notes - Dr Cyril Appleby

Hard work and “carelessness” leads to a world first

As I understand it, Cyril, when you were trying to crystallise this flavonoid cytochrome, which was something that was going to be very hard to do, you were a little bit naughty in terms of your lab duties and went off on a picnic; is that right?

Well, I had found that by acetone fractionation, first stirring in cold acetone to 30% in the steel beaker instead of having to centrifuge the curdy suspension (we did not have a large, refrigerated centrifuge), the precipitate would stick to the sides of the beaker. I would discard this first precipitate because the assay showed no cytochrome b2 and little enzyme activity. Then, at 35% acetone a lovely red oil would separate out. You didn’t even have to centrifuge it; you just had to carefully decant the clear paler red supernatant. This lovely red oil could then be dissolved in a dilute lactate buffer. Lactate proved to be a very important protectant; in general terms, if one can keep a dehydrogenase enzyme reduced, it’s more stable.

I was getting on and on and the purification was getting better and better and already I could see that the lactic dehydrogenase, which was assayed using ferricyanide or cytochrome c as the electron acceptor, was being purified in exact parallel to cytochrome b2 which I was detecting with a microspectroscope or spectrophotometer. I thought, ‘I have got Morton; this dehydrogenase is a cytochrome’.

But one Wednesday I had finished some work trying to adsorb the enzyme on calcium phosphate gel and, by nightfall I’d had enough. I thought, ‘I really am going to go to the staff picnic in the Dandenongs tomorrow’. So I left the rest of the preparation, which had been dialysed to a very low salt concentration with lactate present, under nitrogen in a small tube in the cold room for a day and a half. I came back on the Friday morning and became worried because there was a turbid precipitate in the tube. I thought, ‘This is bad’. But then, when I looked carefully with a hand spectroscope, by moving down the tube I could see a lot of cytochrome c at the top, and down the bottom a different, intense red-pink band which was indicative of cytochrome b2. I thought, ‘My God, I have either done it or I have stuffed it’.

You had the tube with the apparent crystals and Dr Morton became very excited. When did he actually see that?

Morton came in at about five past nine, just before his first lecture. He said ‘Mr Appleby, why are you looking so upset?’ Then I said, ‘Well, I think I have managed to precipitate all of the cytochrome b2’. ‘Mr Appleby, how careless of you. Instead of working yesterday, you went off to a staff picnic’. So he grabbed the tube out of my hand and flicked it and in doing so generated a schlieren, a reflectance pattern characteristic of a crystalline protein. This was something I had not seen before. ‘Mr Appleby, Mr Appleby, crystals, crystals!’ he cried. He grabbed the tube and raced down the corridor to the professor's office. ‘Professor, professor, I have crystallised—ah, ah—we have crystallised the first ever cytochrome’. (In fact I, with Morton’s careful guidance, went on to show that the homogeneous crystalline enzyme contained both flavin and haem prosthetic groups in equal proportion––this was a world first.)

Bucket biochemistry, followed by protein and gene sequencing, shed light on the origin of plant kingdom haemoglobins

Now you became interested in another plant that wasn’t a legume but it had nodules and haemoglobin.

Mike Trinick, a Perth then Canberra CSIRO colleague, had work in New Guinea during the 1960s. One day his then technician said, ‘Dr. Trinick, I have found that this plant Parasponia’—which was an opportunistic Ulmaceae species growing between rows in a new coffee plantation—‘has nodulated roots’. Trinick established that the nodules contained Rhizobium and that they were involved in symbiotic nitrogen fixation. Today, it remains the only situation where a non-legume plant has been found to be associated with Rhizobium in a nitrogen-fixing symbiosis.

Yes; because others like Casuarina have a different bacterium symbiont.

Before Trinick’s discovery of the Parasponia symbiosis, and with haemoglobin known to be widespread in animals but only once in plants (in legume nodules), it was thought that maybe it had got there by a unique act of horizontal gene transmission. Perhaps an insect, via a viral vector, had managed to stick the gene into the plant. I thought–– ‘Now, if I could get hold of Trinick’s Parasponia nodules and show that they contained haemoglobin, we could extract, purify and characterise it and then determine its gene structure’.

So that is when you came to us.

Yes, if that supposed unique act of horizontal gene transmission to a primitive legume could be ruled out, then one had to start thinking about vertical descent of haemoglobin genes between the plant and animal kingdoms.

And we did it!

We did it, yes. (By classical biochemical procedures I extracted and purified much haemoglobin from Parasponia root nodules. Alex Kortt, a Melbourne CSIRO colleague determined its amino acid sequence then Jim Peacock’s group used a gene probe based on this sequence to isolate and characterise the Trema haemoglobin gene. Its similarity to the legume and animal haemoglobin genes was very obvious. We went on to isolate and characterise haemoglobins and/or their genes from other plants, showing that they and animal haemoglobin genes did indeed have a common origin.)

An edited transcript of the full interview can be found at:
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Focus questions

[Students may need access to a science reference book, dictionary or the internet to answer some of these questions.]

• Use reference books and/or the internet to define the following terms:
  • • Legume
    • Plant nodule
    • Haemoglobin
    • Leghaemoglobin
    • Genes
    • Gene transfer
    • Symbiosis
    • Symbiont
    • Nitrogen fixation
    • Rhizobium
  • Dr Appleby used a lactate buffer in his crystallisation procedure. What was its role?
     
  • Draw the chemical structure for acetone. What is its proper chemical name?
     
  • What is a spectroscope and what does it do? Why was it important for Dr Appleby’s research?
     
  • What is nitrogen fixation? Briefly describe why it is important in agriculture.
     
  • What is the name of the enzyme involved in nitrogen fixation?
     
  • Dr Appleby’s work led to demonstration that haemoglobins were present throughout the plant kingdom and that plant and animal haemoglobin had a common genetic origin. After reading the transcript extract, what term is used to describe this kind of gene transmission?
     
  • What is the function of haemoglobin in (a) plants and (b) animals? Where can it be found in each?
     
  • What is the main difference between haemoglobin and leghaemoglobin? Why is leghaemoglobin important?
• Dr Appleby’s research has focused on basic plant research as opposed to applied plant research. Ask students to read ‘Why do basic research?’ (National Institute of General Medical Sciences, National Institutes of Health, USA) and debate the merits of both kinds of scientific research.