AUTOMATED PROTEIN ANALYSIS

297

Accounts of human infection by S. marcescens are

usually accompanied by the advice that this organismshould not be used for the study of experimental infec-tions because it is a potential pathogen. Since it lives inthe bowel and respiratory tract of healthy men it would bea pity to take this advice too seriously. Its appearanceunder the microscope is about the only character it shareswith Salmonella typhi. All the evidence goes to show thatit very seldom produces disease except in a host whoseresistance has been vitiated-and it probably shares thisproperty with every member of the " normal " flora ofthe body.

AUTOMATED PROTEIN ANALYSIS

THE primary structure of proteins, the sequence ofaminoacid residues along the protein chain, is stilldetermined by the time-consuming methods developed bySanger 1 in his work on insulin. The protein is split intosmall peptides by selective hydrolysis, the peptides areseparated, and the sequence of aminoacids in the isolatedpeptides is determined. The aminoacid sequences in thenumerous peptides are then compared and the sequenceof the complete protein is built up in jig-saw puzzlefashion.

The separation and purification of the peptides is verytedious, and losses during this process mean that largequantities of pure proteins are required for sequencedeterminations. W. R. Gray of the California Instituteof Technology suggests 2 that the separation stage may beeliminated. The protein would be split into peptides byspecific enzymatic or chemical cleavage at particularaminoacid residues; the Edman degradation technique 3could then be used to remove aminoacids one by one fromthe amino-terminal end of each peptide in the mixture ofpeptides. At each stage of the Edman degradation amixture of aminoacids will be produced, but by com-parison with degradations on the peptide mixtures fromother cleavage reactions it should be possible to work outa unique sequence for the original protein.Gray calculates that specific cleavage after six amino-

acids, followed by identification of the first ten residues ofthe peptides in each mixture would have been sufficientto determine the structure of ribonuclease, except forposition 24 (asparagine). On a much larger protein, thelight chains of immunoglobulins, the same approach wouldgive the structure of most of the molecule, but fifteenaminoacids would not be determined because one stretchof twenty-five aminoacids would not split. Further

hydrolyses or more extensive degradation of the peptideswould have given the complete sequence.

Specific chemical methods for splitting peptide chainsat the residues methionine, cysteine, tryptophan, tyrosineand histidine,4 lysine,5 and arginine 6 are now available.Some of the factors which limited the usefulness of theEdman degradation can be eliminated, and Edman andBegg have described a

"

sequenator ", a machinewhich splits off the N-terminal aminoacids as thiazolinones1. Sanger, F. Adv. Protein Chem. 1952, 7, 1.2. Gray, W. R. Nature, Lond. 1968, 220, 1300.3. Edman, P. Acta chem. scand. 1950, 4, 283.4. Witkop, B. Science, N.Y. 1968, 162, 318.5. Toi, K., Bynum, E., Novus, E., Itano, H. A. J. biol. Chem. 1967, 242,

1036.6. Goldberger, R. F., Anfinsen, C. B. Biochemistry, 1962, 1, 401.7. Edman, P., Begg, G. Europ. J. Biochem. 1967, 1, 80.

by a series of automatic programmed reactions. The

yield at each step was so good that they were able to takesixty aminoacids from the N-terminal end of humpbackwhale myoglobin before the results became ambiguous.The Edman degradation does not work very well on smallpeptides because they tend to be extracted along with thethiazolinones, but several new techniques promise to

improve this situation.Aminoacid analysers which automatically separate and

estimate aminoacid mixtures are already available and canbe linked to computers which calculate results. The

computer could also cross-check results from differentdigestion procedures to produce the final sequencepattern. If it were possible to regenerate aminoacids

quantitatively from the derivatives formed during theEdman degradation the entire process of sequencedetermination (apart from the initial steps involved in

splitting the protein into peptides) could be automated.The present methods of regeneration of aminoacids fromthiohydantoins are not very satisfactory, and quantitativedeterminations of the thiohydantoins are not very good.The Edman degradation may ultimately be replaced byother degradation techniques-some cobalt complexescan remove N-terminal aminoacids from peptides to givean orange cobalt complex which can be identified chroma-tographically and estimated spectrophotometrically.8The most exciting possibility this new approach offers

is the ease with which one could identify altered portionsof the molecule in proteins whose sequence is known.

Ingram, in his classic experiment on haemoglobin S,9separated the peptide digest by a combination of electro-phoresis and chromatography known as

" fingerprinting ".However, this will give satisfactory results only if themutation alters the mobility of the peptide. Gray’sapproach would immediately indicate which aminoacidsin a tryptic digest differed from those in the normal

protein, and a little further work would pinpoint its exactplace in the sequence.

GREEN-PAPER TIGER

A Special Representative Meeting of the B.M.A. onJan. 30 discussed their Council’s recommendations 10 onthe future administration of the Health Service. The

gathering started in liberal mood, affirming that the singleobject of any changes must be the improvement of theservice to the community and to the patient; but as theseats became harder, language became more intemperate,and opinions more intransigent. It was not long beforephrases such as " by the profession for the profession "," junior partners of the Government ", and " slaves tothe State " could once more be heard in the Great Hallof B.M.A. House. From this agonised debate the Coun-cil’s thirteen recommendations emerged almost unscathed.The B.M.A. has already affirmed its support for the

principle of unification of administration of the HealthService, but in view of the number of delegates whoappeared, in various ways, to oppose this principle, it wassurprising that in the end only six votes were registeredagainst the recommendation. The representatives hadalready, for example, supported the recommendation8. Buckingham, D. A., Collman, J. P., Happer, D. A. R., Marzilli, L. G.,

J. Am. chem. Soc. 1967, 89, 1082.9. Ingram, V. M. Biochim. biophys. Acta, 1958, 28, 539.

10. Br. med. J. Dec. 28, 1968, suppl. p. 78. See Lancet, Jan. 25, 1969, p. 196.