CA2082770A1 - Engineered protein chelates suitable for fluorescent lanthanide (e.g. terbium (iii)) based time resolved fluorescence assays - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A chelator sequence of 12 amino acids can form complexes with luminescent lanthanides such as Terbium and Europium. The complexes display high affinity between chelator and lanthanide and are useful as probes in fluorescent immunoassay.

Description

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The present invention relates to chelator sequences of amino acids, to metals chelated by the chelator sequence, to luminescent lanthanide3 chelated ~o the amino acid sequence and to the use of the luminescent Lanthanide - amino acid complexes in assays.
BACKGROUND OF THE INVENTION
_ In quantitative clinical chemistry, assays that are ba3ed on the use of antibodies, to give selectivity, and radioiso-topes, to give sensitivity, dominate. The procedure is known as radio-immunoassay (RIA). However, there has been increasing effort to eliminate the use of radioisotopes because of potential health hazards in their use, expense of disposal, and limited shelf-life of reagent~. In the search for rapid, sensitive, and non radio-isotopic assay methods, the u~e of fluorescence based procedures, i.e., fluorescent immunoassay (FIA), has become of great interest.
The first fluorescence probes suffered seriou-q limita-tions to sensitivity owing to interference from natural fluor-eRcence from various compounds in biochemical samples such a~
blood serum. Filters have been used, but even then these fluores-cence methods are not as ~ensitive as radio-immunoassays. The use of pulsed-light source time-resolved fluorometers, as described in Canadian Patent No. 1~082~106r has been applied to increase great-ly the sensitivity of clinical diagno~tics so that the FIA method 2~

can compete with the RIA method. A review of fluoroimmunoassay methods appears in a paper entitled "Immunoassays with Time -Resolved Fluorescence Spectroscopy: Principle~ and Applications", by E.P. Diamandis in Clinical Biochemi3try, Volume 21, pp. 134-150, June 1988, and other immunoa say methods are the subject of references listed at the end of the review.
In order to benefit from the inherent sensitivity of time resolved principles, a f1uorescent label to substitu~e for radioisotopes must be used. The luminescent lanthanides, for example Europium, Terbium, Samarium and Dysprosium are of interest, Terbium and Europium being of primary interest. These have been used with organic chelates, for example EDTA based or diXetone ligands.
Many organic chelators of luminescent lanthanides have been synthe~ized; see for example U.S. Patent Nos. 4,374,120;
4,637,988 and 4,772,563. These chelators were based on oxygen atoms or nitrogen atoms or both as donor atoms and are, for exam-ple, diketonec or dicarboxylates. Some chelating agents enhance the emission of the lanthanides by energy transfer ~rom the organic portion of the chelating ayent following irradiation by a light ~ource, for example UV lamp or laser. Whether enhancement occurs depends upo~ the overlap of the fluorescence and/or phosphorescence spectrum of the organic chelating agent, or chromophore, and the absorption spectrum of the lanthanide, i.e., whether Forster Resonance Energy Transfer or Dexter exchange can take place between the chromophore and the lanthanide. Hence, ideally, a particular chromophoric chelating agent should be selected ~or a particular lanthanide.

2~ 7 1~

Various disadvantages accompany use of the diketone and dicarboxylate chelating agents. For instance, all lanthanide~ can have a coordination number up to 9, but more usually have a co~
ordination number of 6 or 8, i.e., the lanthanide will, theoreti-cally, bind to three or four bidentate chelating agents, respect-ively. Many of the chelating agents are bidentate so that to chelate fully an ion with a coordination number of 6 three mole-cules of bidentate chelating agent would be required to bind to the ion. With bidentate ligands this rarely happens; usually only one or two molecules of chelating agent bind to the ion. This has two di~advantages. The strength of the attachment between the central atom and the chelating agent, i.e., the affinity constant, is less than it would be if all coordination Rites were occupied by the -~ame chelating agent, and hence the sensitivity as a ~robe in FIA is less. The coordination sites not occupied by chelating agent are frequently occupied by water. Water has a quenching effect on luminescence, which again reduces sensitivity.
SUMMARY OF THE INVENTION
In one aspect, the present invention provide~ a chelator sequence selected from the group consisting of ~equences of twelve amino acids containing the following amino acids:
SEQ ID ~O:l:

Asx Xaa Asx Xaa Asx Xaa Xaa Xaa Glx Xaa Glx Glx and sequences of the said amino acids in which one or more of the said amino acids is bonded to a chromophore.
This amino acid sequence can also be expressed, using the one letter notation for amino acid sequences proposed by IUPAC-IUB~ as follows:
B X B X B X X X Z X Z Z

The number indicates the residue number in the chelator sequence. Residues 1, 3, 5, 9, 11 and 12 are residues that can donate electrons to the chelated metal ion.
In another aspect, the invention provides a complex comprising a luminescent lanthanide and a chelator ~equence selec-ted from the group consi~ting of sequences of twelve amino acid containing the following acidR:
SEQ ID NO:2:

Asx Xaa Asx Xaa Asx Xaa Xaa Xaa Glx Xaa Xaa Glx and sequences of the said amino acid~ in which one or more of the said amino acids is bonded to a chromophore.
This sequence can be expressed in ~ingle letter notation as follow~:

B X B X B X X X Z X X Z

It is preferred that in this complex position 11 of the amino acid 3equence is occupied by Asx.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The following Table 1 lists amino acids and the corres-ponding one letter symbol~ and three letter symbols proposed to IUPAC-IUB.

r Table I

= ~ . . .. ~ .. _ _ . _, _ _ _ , One-Letter Three-Letter ~ymbo1 symbol Amino acid ~ _ _ _ _ . . . _ _ A Ala Alanine B Asx Aspartic acid or asparagine C Cys Cysteine D Asp A~partic acid E Glu Glutamic acid F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isole~cine ~ Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine ~ Arg Arginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan X Xaa Unknown or "other"
Y Tyr Tyrosine Z Glx Glutamic acid or glutamine In one preferred embodiment of the invention the chelator sequence i8 as follows:
SEQ ID NO:15:

A~p Xaa Asn Xaa Asp Xaa Xaa Xaa Glu Xaa Glu Glu In a more preferred embodiment the chelator ~equence is as ~ollows:
SEQ ID ~0:16:

ABP Xaa Asn Xaa Asp Xaa Trp Xaa Glu Xaa Glu Glu A preferred complex comprises a luminescent lanthanide and a chelator sequence as follows:

~C~ r ~

SEQ ID NO:17:

Asx Xaa Asx Xaa Asx Xaa Xaa Xaa Glx Xaa Xaa Glx In a more preferred complex the chelator sequence i~ as follows:
SEQ ID NO:18:

Asx Xaa Asx Xaa Asx Xaa Xaa Xaa Glx Xaa Glx Glx wherein Xaa in the 7-position is Trp, Tyr or Phe or a derivative of Trp, Tyr or Phe, or Cys derivatized by a chromophore.
In another embodiment the complex comprises a chelator sequence as follows:
SEQ ID NO:l9:

Asp Xaa Asn Xaa Asp Xaa Trp Xaa Glu Xaa Xaa Glu Examples of specific preferred chelator sequences of amino acids in accordance with the invention include the follow-ing:
SEQ ID NO:3:
Asp Lys Asn Ala Asp Gly Trp Ile Glu Phe Glu Glu SEQ ID NO:4:

Asp Lys Asn Ala Asp Trp Gly Ile Glu Phe Glu Glu 1 ' 5 10 SEQ ID NO:5:

A~p Lys Asn Ala Asp Ala Trp Ile Glu Phe Glu Glu SEQ ID NO:6:

Asp Lys Asn Ala Asp Gly Trp Ile Glu Trp Glu Glu SEQ ID NO:7:

Asp Lys Asn Ala Asp Ala Trp Ile Glu Trp Glu Glu 1 5 1~

~ J~

. SEQ ID N0:8:

Asp Lys Asn Ala Asp Trp Gly Ile Glu Trp Glu Glu SEQ ID N0:9:

Gly Asp Lys Asn Gly Asp Gly Trp Ile Glu Phe Glu Glu Leu SEQ ID N0:10:

Gly Asp Lys Asn Gly Asp Gly Tyr Ile Glu Phe Çlu Glu Leu 1 5 . 10 SEQ ID N0~

Gly Asp Lys Asn Gly Asp Gly Phe Ile Glu Tyr Glu Glu Leu SE9 ID No. 12 Gly Asp Lys Asn Gly Asp Gly Tyr Ile Glu Trp Glu Glu Leu SEQ ID ~0:22:

Asp Lys A-~n Ala Asp Gly Cy8 rle Glu Phe Glu Glu SEQ ID N0:20:
Gly Asp Lys Asn Ala Asp Gly Cys Ile Glu Phe Glu Glu Leu SEQ ID N0:21:
Ser Leu Val Ala Leu Asp Asn Asn Ala Asp Gly Cys Ile Glu Phe Glu Glu Leu Ala Thr Leu Val Ser The sequence of twelve amino acids constitutes one mole-cule, or portion of a molecuLe, that can satisfy all the coordina-tion sites of an ion that has a coordination number of 6, 7, 8 or 9 i.e. the chelator sequence forms a hexadentate to nonadentate ligand. Particularly when the amino acid sequence is part of a large peptide chain, as di~cussed below, the amino acid sequence and the ion are strongly bound; affinity constants greater than lOg and sometimes greater than 1011 are obtained. ~urthermore, as S~ ~

more coordination sites of the ion are occupied less water binds to the metal and water quenching of luminescence is reduced.
Water quenching can sometimes be reduced further by use of a detergent molecule which displaces water in the coordinating cage around the metal atom. A suitable detergent for this purpose is tri-n-octylphosphine oxide, known as TOPO.
The amino acids not specified in the chelator sequence, i.e. those in the 2, 4, 6, 7, 8 and 10 positions are selected with regard to the likelihood that a particular amino acid will assist in binding, or at least will not interfere with binding. For instance proline may prevent the amino acid sequence from adopting the conformation neceRsary for chelating, so use of proline is not preerred. If too many negatively charged atoms are present in the chelator sequence electrostatic repulsion may affect the bind-ing affinity, so the chelator sequence should not contain too many aspartic or glutamic acid moieties, or should be balanced by lysine arginine or histidine moieties.
If the chelator sequence i8 to be used with a lumin-escent lanthanide in, for example, FIA, then the unspecified amino acids can also be selected with regard to the desired luminescence effect. To enhance luminescence it is desirable to include in the chelator sequence an amino acid that includes an aromatic ring.
The order of preference of such acids i~ tryptophan, then tyrosine and then phenylalanine. For instance tryptophan is a chromophore whose luminescence 4pectrum overlaps with the absorption spectrum of Terbium. Therefore energy transfer from tryptophan to Terbium occurs and enhanced luminiscence of Terbium is observed. Conse-quently if Terbium is the lanthanide being used tryptophan is to be preferred to, say, tyro~ine, as an amino acid to occupy un-specified positions. When luminescence i8 required it is prefer-red that tryptophan occupies position 7 of the 12 amino acid sequence.
The amino acids that occupy the unspecified positions will normally be selected from those amino acids that appear in naturally occurring peptides or protein~. It is possible to use other closely related amino acids, however. Amino acids that differ from tho~e that occur in nature by replacement of a ring methine group by a nitrogen atom, by replacement of a hydroxy group by an alkoxy group, by insertion of an alkyl group in place of a hydrogen atom attached to a basic nitrogen atom or by inser-tion of an alkoxy group or a halogen atom, preferably a fluorine atom, in place of a hydrogen atom on an aromatic ring, can be used. Mention i8 made, for example, of 7-azatryptophan, 5-methoxytryptophan, 4-fluorotryptophan and 4-methoxytyro ine.
The chelator sequence of twelve amino acids can be used alone but it i9 preferred that the sequence forms part of a larger molecule. There is known a family, defined by genetic lineage, of high affinity metal-binding proteins which includes the much studied calcium binding proteins calmodulin, troponin C, parval-bumin, calbindin 9K and oncomodulin. These proteins contain one or more sites, usually two to four sites, which are loops of amino acids situated between alpha-helices. The loops usually bind strongly to calcium. For instance parvalbumin has two calcium binding -~ites and calmodulin has four calcium binding sites per molecule. Oncomodulin has three loops known a~ the AB, CD and EF
loop~. Of these the CD and EF loops bind calcium strongly, but the AB loop does not. It is believed that the AB loop at one time had the ability to bind calcium but for some reason that ability has been lost.
These calcium binding proteins will bind Terbium (III) and other luminescent lanthanides with affinity constants up to 103 or more times greater than they bind calcium. In preferred embodiments of the invention amino acids of a loop of a c~lcium binding site in a calcium-binding protein are replaced by a chelator sequence of twelve amino acids in accordance with the present invention. For instance oncomodulin has the CD calcium binding site between amino acids 50 and 63 and the EF site between amino acids 89 and 102. It i5 preferred to replace the amino acids of one or both of these sites by a chelator sequence of amino acids in accordance with the invention. Another site in oncomodulin which is preferred for substitution by a chelator sequence of the present invention i8 that of the A~ loop between amino acids lS and 28.
Some of the amino acids of the sequence of the invention will be present in the loops in the calcium binding sites of the naturally occuring binding protein and others will not be pre3ent, but will have to be inserted in place of other amino acids that, in nature, occur at that particular poqition. The number of amino acid~ inserted should equal the number of amino acids removed, so that the general conformation at the binding ~ite, i.e. a loop flanked by alpha-helices, is maintained. Although it may not be necessary to replace all twelve amino acid~ in a loop to obtain a sequence in accordance with the invention, for the sake of sim-plicity of description and definition reference will be made to ~" ~ J ~

replacing naturally occuring 12 amino acid sequence by 12 amino acid sequences of the invention.
The chelator sequence of amino acids can be used as such or as part of a larger molecule, as indicated above. As it is composed of amino acids it can readily be attached to a protein by well known methods and then chelated with a luminescent lantha-nide, to provide a fluorescent marked protein. For instance, at the one end of the amino acid sequence there will be an amino nitrogen atom and at the other end of the equence there will be a carboxyl group. These can be used to bind to other molecules.
Some amino acids contain functional groups which can be used to crosslink the amino acid sequence to the other mo]ecules. For example lysine can crosslink via an amino group, cysteine can crosslink via a sulphur atom, serine can crosslink via a hydroxy group and the dicarboxylic amino acids can crosslink via the carboxyl group of the ~ide chain. If necessary an amino acid able to croYslink can be attached at the N-end or the C-end of the 12 amino acid sequence.
In addition to the calcium binding proteins mentioned above, other proteins can be modified, by chemical synthesis or by genetic engineering, to incorporate a chelator ~equence of amino acids in the molecule.
The chelator sequence of twelve amino acid-~ can be made by chemical synthesis or by genetic engineering. When the twelve amino acid chelator sequence constitutes the whole or a major part of the molecule the method of chemical ~ynthesis is preferred.
This method may also be preferred when the sequence i5 to be attached to the end of a peptide chain, for instance when attached $~ 7 ~

as a marker to a hormone, an antibody or a tumour marker. When the sequence is to be incorporated at one or more intermediate points of a peptide or protein then genetic engineering may be the preferred method. Expression systems, for example bacterial and yeast expression systems, for at least 30me of these proteins are known and mutant proteins having amino acids selectively exchanged can be obtained by site directed mutagenesis.
Luminescent complexes of the invention can be used as markers or probes in various assay methods to detect the presence of, for example, antibodies, antigens, hormone~, en~ymes, carbo-hydrates, DNA ~equences, etc and even lanthanides themselves.
Thus the assay method can involve forming a complex or chelate of the amino acid sequence of the invention and a luminescent lan-thanide, and o~erving the fluorescence of the complex formed.
The analyte can be the amino acid sequence it~elf, it can be a molecule bound to the amino acid sequence or to a molecule con-taining the amino acid sequence or it can be the luminescent lanthanide itself. The detection can be qualitative or quantita-tive. When the chelator sequence of twelve amino acid~ form the total molecule the affinity for the lanthanide is usually lower than when the chelator sequence i9 part of a longer peptide or protein chain. Complexes with low affinity can be used for qualitative determinations but complexes with high affinity are preferred for quantitative determinations. One assay method is a sandwich assay method in which an antibody i9 immobilised, for instance by binding to a glass surface, or the like. Antigen, whose presence is to be detected, is passed over the surface and binds to the immobilised antibody. Thereafter a further antibody, 2t~ ,7~

which is ?abelled by being attached to a fluorescent complex of the invention, is passed over the ~urface. The further labelled antibody binds to the bound antigen~ ExcesA labell0d antibody is washed away. Thereafter the fluorescence spectrum of the material bound to the glass surface i~ obRerved and this serves as a quali-tative or quantitative indicator of the presence of the antigen.
The chelator sequences of the invention can readily be chelated to metals such as the lanthanides by known methods. It is usually desirable to chelate at a p~ below 7 and if a buffer is to be used it is preferred that it contain nitrogen atoms in preference to oxygen atoms; a preferred buffer is piperazine-N,N'-bis~2-ethane-~ulfonic acid], known as PIPE5. The ionic ~trength should not be too high, preferably not greater than about 0.1 M.
Although the chelator amino acid sequences of the inven-tion are particularly u~eful for chelating luminescent lanthanides for use as probes or markerY for fluorescence immunoassays, they can be used to chelate metals other than luminescent lanthanides and they can be used in a~ays other than fluorescence immuno-a~says. For example, they can be used to bind calcium. It is possible to immobilise a chelator aequence of amino acids, or a peptide containing a chelator sequence of amino acids, on a suit able substrate such a~ polyacrylamide beads. If a solution containing calcium is pas~ed over these beads the calcium ions will be chelated by the amino acids and im bilised with them, so that the ~olution is freed of calcium.
The chelator sequence can also be uYed to assay for lanthanides. Terbium displays luminescence but a large quantity %f.~. g~

of Terbium is normally required, only a low luminescence effect is observed even at Terbium concentrations as high as 0.25 M. As i8 demonstrated in an example below, addition of a particular chelator sequence of the invention (having ability to enhance luminescence of Terbium) to a solution of Terbium resulted in Terbium being detected at a concentration of 7xlO-1 mM.
The chelator sequence of the invention differs in effect from the calcium-binding loops found in nature. The naturally occurring loops can both bind and release calcium ions and after a calcium ion has been released by a naturally occurring loop the loop is available again to bind a calcium ion. The naturally occurring loop can be switched from calcium binding to calcium releasing. In contrast, the amino acid ~equences of the invention do not readily release bound calcium ions; to release the calcium ions it is necessary to change the conformation of the sequence by protein denaturation for example by heat, acidification, alkalini-sation, high salt concentration, etc.
The chelator sequence of the invention or a luminescent complex of the invention can be attached to other compounds of interest such as avidin or thyroglobulin, and used, for example, in amplification systems usin~ biotin.
The chelator sequence or the luminescent complex can also be bound to a molecule containing another active site for use in assays. For instance a complex of Terbium and an amino acid sequence of the invention may be bound as part of a molecule that also contains e.g. Europium bound to an organic non-proteinaceous chelating agent. With such a reactant it is possible to conduct C~ J i 9i l7 one test to detect two analytes.
In one embodiment of the invention an amino acid of th~
twelve amino acid sequence is covalently bonded to a chromophore.
If the chelator sequence is then used to chelate a fluorescent metal there can occur energy trans~er from the chromophore to the metal, to enhance luminescence.
The amino acids of the twelve amino acid sequence that chelate directly to a metal atom are the acids that occupy posi-tions 1, 3, 5, 7, 9 and 12 of the 3equence. It is desirable that the chromophore shall be as closed to the ~etal as possible, to facilitate energy transfer, so it i9 preferred that a chromophore i8 covalently bonded to one or more of the acids in these posi-tions. A chelator sequence having a chromophore attached to one or more of the positions 2, 4, 6, 8, 10 and 11 is still within the scope of the invention, however.
The invention requires that positions 1, 3 and 5 shall be occupied by aspartic acid or asparagine. These compounds have side chains containing a carboxyl group or an amide group, respectively. When these chelate, an electron pair from the carbonyl oxygen atom of the side chain is used to ~orm the chelat-ing bond to the metal atom. The invention requires that positions 9 and 12 shall be occupied by glutamic acid or glutamine, which again have side chain~ containing a carboxyl group or an amide group, respectively. When these chelate, it is again an electron pair from the carbonyl oxygen atom of the ~ide chain that is used to form the chelating bond~ In order to locate a chromophore close to the metal, it is preferred to use in position 1, 3 or 5 2 ~` ~3 sç~ 7 r~ ~

aspartic acid or asparagine in which the OH group or the NH2 group respectively, has been replaced by a chromophore. Alternatively or additionally, there may be used in position 9 or 11 glutamic acid or glutamine in which the OH group or the ~H2 group has been replaced by a chromophore. The chromophore is thus attached to the carbon atom of the carbonyl group whose oxygen atom is supply-ing the electron pair of the chelating bond.
Suitable chromophores for attachment to the carbonyl group of the side chain of aspartic acid, asparagine, glutamic acid or glutamine include derivatives of pyrene coumarin ~alicylic acid, benzophenone and dimaleimidylstilbene and the like. Such compounds are known to persons 3killed in the art. Sequences containing chromophores can be prepared by well known methods of peptide synthesis, using in place of one or more of the aspartic or glutamic moieties a chromophoric derivative of the aspartic or glutamic moiety. These synthetic methods permit the location of the chromophore-bearing acid at a selected position or positions in the sequence.
Chromphore-bearing aspartic derivatives for use in posi-tion 1, 3 or 5 of the sequence, and chromophore-bearing gLutamic derivatives for use in position 9 or 12 can be obtained by a condensation reaction between a chromophore and the ~ide chain of the aspartic acid, asparagine, glutamic acid or glutamine. The chromophore ~oiety will contain one or more aromatic rings, usual-ly fused together, for example naphthalene, anthracene, phenan-threne, pyrene or coumarin moieties. Attached to the aromatic ring will be a functional group that can react with the amino acid ) r.~

ide chai~ to form, for instance, an ester or amide. E~amples of compounds that can be condenRed with the aspartic acid, aspargine, glutamic acid or glutamine include N~ pyrenyl)-hydroxyactamide, ~-(l-pyrenyl)-amino-acetamide, 7-diethylamino-3-~(4'-hydroxy-acetylamino)-phenyl-4-methylcoumarin, 7-diethylamino-3~((4'-amino-acetylamino)-phenyl-4-methylcoumarin, hydroxyacetamidosalicylic acid, aminoacetamidosalicylic acid, l-pyrenemethyl hydroxyacetate, l-pyrenemethyl aminoacetate, benzophenone-4-hydroxyacetamide and benzophenone-4-aminoacetamide.
The acids that occupy the 2, 4, 6, 7, 8, 10 and 11 posi-tion-q are not limited to aspartic acid, asparagine glutamic acid and glutamine. The acids in these positions can be bonded to a chromophore, bonding at po~ition 7 being preferred. The amino acid to which the chromophore is bonded should contain an extra functional group, in addition to the carboxyl group and the amino group of the amino acid. This extra functional group is used to bond covalently the chromophore to the amino acid. Acidq that can be used include cysteine, lysine, methionine, threonine and arginine, of which cysteine in position 7 is preferred.
So that there i~ ~peci~icity in the polnt of attachment of the chromophore, it i~ desirable that the amino acid appears only once, at the desired position, in the chelator sequence.
If cysteine is present only in the 7-position then a chromophore that reacts selectively with cysteine will attach only at the 7 -pO8 i tion.
By way of example, there is described the obtaining of a pro~ein including a twelve amino acid chelator sequence of the ~ f~ 3 ~

invention, in which position 7 is occupied by cysteine bonded to a chromophore. By cassette mutagenesis a protein is modified to replace a naturally occurring twelve amino acid equence of the invention that has cysteine at position 7. The obtained, modified protein is then reacted with a chromophore that will bond only to cysteine. There is therefore obtained a protein that includes a twelve amino acid ~equence of the invention having a chromophore bonded to cysteine at position 7 of the twelve amino acid sequence. To achieve ~pecificity of attachment of the chromo-phore, it may be neces~ary or desirable to remove an amino acid that appears in the protein outside the twelve amino acid sequence and to replace it by an amino acid that i9 non-rsactive with the chosen chromophore. For instance, oncomodulin has a cysteine moiety at position 18. When the CD loop of oncomodulin was re-placed by a twelve amino acid sequence of the invention having cysteine at po~ition 7 of the sequence, as described in Example XI
below, the cysteine at position 18 of oncomodulin was replaced by valine. Consequently there was in the obtained protein only one cysteine moiety.
Chromophores that can react with cysteine, or other mercapto-containing amino acids, include N-(l-pyrenyl)-iodoacet-amide (PIA), 7-diethylamino-3-((4'-iodoacetylamino)-phenyl)-4-methylcoumarin ~DCIA), iodoacetamido~alicylic acid (IASA), 1-pyrenemethyl iodoacetate (PMIA), 4,4'-dimaleimidylstilbene (DIMS) and benzophenone-4-iodoacetamide (~PIA). An iodine-containing chromophore can be reacted in known manner with the mercapto-containing amino acid in aqueous solution at a slightly alkaline 7 ~ !J',~

pH, about pll 8. The solution can be buffered, for example with a buffer composed of 150 mM KCl and 10 mM Tris. A condensation reaction takes place between the iodine-containing chromophore and the acid. The reaction is illu~trated with reference to N-(l-pyrenyl)iodoacetamide and cysteine, a follows:

[ ~ NH-C-CH2-I +HSCH2CHCOOH
\

1~1 `/

~ NH-C-CH2-S-CH2-CHCOOH + HI
W

Cysteine will react under -~imilar conditions with the maleic double bond of dimaleimidylstilbene by adding to the double bond, resulting in formation of a covalent bond between the sulfur atom and a carbon atom of the double bond.
Chromophores for attachment to the chelator sequence can be considered to be composed of a chromophoric moiety and a linker moiety. When the chromophore is to be linked with a mercapto group, as with cysteine, a preferred linker moiety contains an iodoacetyl group that reacts as described above. If the chromo-phore is to be attached to a functional group other than a mercap-to group the linker moiety will be different. For instance, for attachment to an amino group as with lysine or arginine, % ~ i 7 ~ ~

an i othiQcyanate, succinimide or sulfonyl halide linker moiety can be used. For attachment to a hydroxy group as with serine or threonine, an acyl nitrile or acyl azide linker moiety can be used. Chromophores composed of a chromophoric moiety and such linker moieties are known and are commercially available from, for instance, Molecular Probes, Inc. of Eugene, Oregon, USA.
It has been ~ound, and is demonstrated below, that bind-ing of both Tb3+ and Eu3+ to a PIA-14mer of the invention results in significant enhancement of luminescence. Similarly, Eu3+
luminescence i8 enhanced by a DCIA-14mer of the invention.
A preferred chelator sequence i8 a 23mer of following structure:
SEQ ID NO:21:

Ser Leu Val Ala Leu Asp Asn Asn Ala Asp Gly Cys Ile Glu Phe Glu Glu Leu Ala Thr Leu Val Ser In this sequence, the twelve membered chelator sequence is ~lanked by two stretches of residues that have a tendency to alpha-helix formation, modelling the naturally ocurring helix-loop-helix motif in oncomodulin. This sequence was covalently bonded to DCIA, chelated with Eu3+ and luminescence was measured. A l~mer sequence was covalently bonded to DCIA, chelated with Eu3+ and luminescence was mea~ured. It was found that the 23mer gave a greater enhancement of luminescence, suggesting that the short alpha-helical portions played a stabilizing role in binding the lanthanide.
Another preferred sequence of the invention is a twelve chelator sequence inserted, by cassette mutagenesis, into onco-modulin in place of the naturally occurring CD loop. This sequence is as follows:
SEQ ID NO:22:

Asp Lys Asn Ala Asp Gly Cyq Ile Glu Phe Glu Glu Oncomodulin modified to contain this ~equence in place of the natural CD loop i8 referred to aY construct 3. It was found that, with DCIA construct 3, Eu3+ could be detected at a concentration as low as 10-1 moles/litre. Filling of both metal binding site~
of the modified oncomodulin by Eu3+ could be detected.
Construct 3 was also covalently bonded to the chromo-phore IASA, and found to enhance Tb3+ luminescence. Again, fil-ling o~ two metal binding ~ites could be detected. Using a fixed concentration of Tb3+(2 M), 10-1OM of IASA-con~truct 3 could be detected.
The invention i9 further illu~trated in the following examples and accompanying figures. Example I uses chelator ~equences made by chemical synthesis and Examples II to VI use sequences made by genetic engineering of oncomodulin and show procedures used to produce oncomodulin and to produce modified oncomodulin~, parbicularly oncomodulin which has been modified to replace the natural amino acid sequence rom 51 to 62 of oncomodu-lin by the sequence identified above as SEQ ID NO:3 to form a modified oncomodulin (hereafter Conqtruct I), in accordance with the invention.
Reference is made to drawing~, of which:
Figure 1 is a purity profile of a peptide (by reverse 2f~3~J~7 ~

phase chromatography on a PepRPC HR5/5 Pharmacia column);
Figures 2a, b, c and d repre~ent the fluorescence spec-tra of peptide of Example I when measured in the presence and absence of exces~ Terbium, Figure 3 illustrates the strategy in constructing a plasmid for expression of oncomodulin, Figure 4 illustrates the nucleotide equence of the junction between the TAC promoter and the oncomodulin sequence of a new plasmid;
Figure 5 illustrates the correct plasmid sequence for expre3sion of oncomodulin mutant Glu 59. The DNA sequence of the mutant Glu 59 and the native Asp 59 is shown where the native sequence for the 59th amino acid has been altered from GAT to GAG;
Figure 6 illustrates the W spectra of bacterially expre~sed oncomodulin and oncomodulin from rat hepatoma;
Figure 7 demonstrates the antigenic cross-reactivity of the bacterially expressed oncomodulin and that of oncomodulin from rat hepatoma Figure 8 illustrates the fluorescence excitation spec-trum of apo native Glu 59 bacterially expressed oncomodulin and its response to the addition of calcium;
Figure 9 shows the EcoRV to Sst I fragment of plasmid pGEM-TAC-ONCO and of pla~mid pGEM-TAC-ONCO-CI;
Figures 10 and 11 are graphs, on different scales, illu-~trating the fluorescence at 545 nm of complexes of Terbium bound to oncomodulin and to oncomodulin mutants, particularly an C~ 7 ~ ~

oncomodulin modified to contain (Construct I) between amino acids 50 and 63 of native oncomodulin, Figure 12 iq a graph howing the fluorescence of tyro-sine when the tyrosine is part of an amino acid sequence bound to Terbium;
Figure 13 is a graph showing the fluorescence of trypto-phan when the tryptophan i5 part of an amino acid ~equence bcund to Terbium:
Figure 14 is a graph showing relative fluorescence intensity at different concentrations of Terbium bound to oncomod-ulin modified in accordance with the invention (Construct I); and Figure 15 ~hows the elution profile of the Trp 57 onco-modulin on a Sephadex G50 column.
Figures 16A and 16B are graphs showing the effect of addition of Tb3+ (squares) or Eu3+ (triangles) to a PIA-14mer on the luminescence of added lanthanide.
Figure 17 i3 a graph showing the effect of different metal ions on light Qcattering from a PIA-14mer, using Tb3+ (tri-angles), Eu3~ (circles) and Ca2+ (squares).
Figure~ 18A and 18~ are graphs showing the effect of Eu3+ addition to a DCIA-14mer on Eu3+ Luminescence.
Figur~ 19 i8 a graph ~howing the effect on Eu3+ lumines-cence of Eu3~ addition to DCIA-14mer (open triangles) or DCIA-23mer (~illed triangleq).
Figure 20 is a graph showing Eu3+ luminescence measured after addition at various concentrations to 200~L 1.5~M DCIA-construct 3 in 150 mM KCl, lO~M PIPES, pH 7Ø

~ ~ 7, ~ 3 Figure 21 is a graph showing the effect on Eu3~ lumines-cence of Eu3+ addition (lO~M stock) to DCIA-construct 3 (l~M) in 200~L of l50mM KCl, 101D~S PIPES, pH 7 . 0.
Figure 22 is a graph showing the effect on Tb3~ lumines-cence of Tb3~ addition (lO~M stock) to IASA-construct 3 (l~M3 in 200~L of 150mM KCl, lOmM PIPES, pH 7Ø
Figure 23 i8 a graph ~howing the effect on Tb3+ lumines-cence of holding ~Tb3+] = 2~M and varying level of IASA-construct 3 in 200~L of 150mM KCl, lOmM PIPES, pH 7Ø
Example I:
This example reports studies with peptides similar to the Construct I binding loop and the CD binding loop of oncomod-ulin and demonqtrates binding efficiency of the Contruct 1 sequence and preference of the aromatic amino acid position for efficient Re~onance Energy Transfer.
Six peptide of 14 amino acid~ were prepared by ynthe-tic methods. Of these, peptide~ 2 to 5 (see below) are similar to Con~truct I. Peptides 6 and 7 are not similar to Construct I, but they do demonstrate the effect of an aromatic amino acid at posi-tion 7.
The protected peptide resins were aynthesized u~ing a p-methyl-benzhydrylamine resin (100-200 mesh, 04-08 meq/g) and N-~-tertiarybutoxy-carbonyl(t-boc)amino acids by the method of simultaneou3 multiple peptide synthe~is (SMPS) developed by Houghten (Reference 15, hereby incorporated by reference) from the original solid phasP peptide synthesi~ method of Merrifield (Reference 16, hereby incorporated by reference). The peptides ~ , 2 7 ~
were cleaved hy the conventional hydrogen fluoride/anisole proce~
dure (Reference 17). A typical purity profile (by reverie phase chromatography on a PepRPC HR5/5 Pharmacia column) ~or a peptide is shown in Figure 1. The column was eluted with a gradient of 0.1~ TFA/H20"(Solvent A~ and 0.1% TFA/CH3CN (50lvent B). 200 ~g of peptide 3 ~as injeeted in 25 ~1 of Solvent A. The gradient pxoceeded from 0-60% in 20 minutes at a flow rate of 0.7 ml/min.
Detection was made by a Pharmacia W -M monitor at 214 nm. This elution profile demonstrate3 that the peptide is approximately 95 pure.
Peptides ~imilar to Construct I:
Peptide 2 SEQ ID NO:9:

Gly A p Lys Asn Gly Asn Gly Trp Ile Glu Phe Glu Glu Leu Peptide 3 SEQ ID NO:10:

Gly Asp Lys Asn Gly Asp Gly Tyr Ile Glu Phe Glu Glu Leu Peptide 4 SEQ ID NO:ll:

Gly A~p Lys Asn Gly Asp Gly Phe Ile Glu Tyr Glu Glu Leu Peptide 5 SEQ ID NO:12:

Gly Asp Ly~ Asn Gly Asp Gly Tyr Ile Glu Trp Glu Glu Leu Peptides similar to CD binding loop of Oncomodulin:
Peptide 6 SEQ ID NO:13:

Gly Asp Asn Asp Gln Ser Gly Tyr Leu Asp Gly Asp Glu Leu ~ , 2 ~ ~ ~

Pept lde 7 SEQ ID NO:14:
Gly Asp Asn A3p Gln Ser Gly Trp Leu Asp Gly Asp Glu Leu The peptides 2-5 were 14mers where residues 2-13 had a similar sequence as the 12 amino acids which were incorporated into Construct I. A glycine in position 6 is qubstituted for an alanine in the Construct I protein ~equence.
The tryptophan re~idue (position 7) in pept ide 2 corre-sponds to tryptophan 57 in the Con~truct I protein. The peptides each have a glycine at the N terminal end and a leucine at the C
terminal end. The peptides 3, 4 and 5 are variant~ of peptide 2.
Peptide 6 corresponds to the CD binding loop of oncomodulin and peptide 7 ha~ a tryptophan residue substituted for the tyrosine, corresponding to tyrosine 57 in the native oncomodulin.
Figure 2 represents the fluorescence spectra of each peptide in the presence and absence of excess Terbium. These were measured as indicated in Example II below.
Figure 2a show~ that peptide 2, which i~ analogous to the binding loop inaerted into Construct I, binds Terbium, and tryptophan 7 transfers energy to the bound Terbium resulting in a Terbium lumineqce~ce at 545 nm. The tryptophan fluorescence is also quenched on binding Terbium. In the case of peptide 3 (Figure 2b) which was the ~ame as peptide 2 except a tyrosine residue wa~ substituted in place of tryptophan 7, again Terbium luminescence could clearly be observed. Peptide~ 4 and 5 (Figures 2c, d) ~how that location of an absorbing chromophore in position 10 (corresponding to rejidue 60 in oncomodulin), resulted ~ s~7;7 ~

in a poor,energy transfer to the Terbium when bound to the pep-tide. It i~ assumed in this latter statement that these peptides bound Terbium with ~imilar efficiencies as peptide 2.
Peptide 7 (Figure 2a) has a tryptophan located in posi-tion 7 and the balance of the ~equence i~ similar to the binding loop of native oncomodulin' ~ CD site. Only a low intensity of Terbium luminescence was observed. Peptide 6 (Figure 2b) has a tyrosine located in po~ition 7, and also ~hows the same low level of Terbium luminescence. This probably indicates that peptides 6 and 7 do not bind Terbium with the same affinity a~ the Con-struct I type peptides, 2-S.
Studies Involving Oncomodulin Until recently oncomodulin has been available only in limited quantities from the rat Morris hepatoma 5123 and other tumours. We report here a bacterial e~pression sy~tem for onco-modulin which permits the isolation of oncomodulin in large quantities and also permits site directed mutagenesis to isolate oncomodulin modified to contain chelating sequences of amino acids in accordance with the invention.
Construction of Expres~ion Plasmids: A bacterial vector for expres~ion of,oncomodulin wa~ constructed from the coding ~equence of an oncomodulin cDNA joined to the TAC promoter (References 1, 2; see below) (TAC promoter Genblock, Pharmacia P~) inserted in the plasmid pGEM-l (Promega Biotec). The oncomodulin coding sequence wa~ obtained from pONCO-4, a plasmid containing the entire oncomodulin sequence as well as 73 nucleotides of the 5'-non coding sequence and 253 nucleotide~ of the 3'-non coding 2 t~
sequence of the oncomodulin messenyer RNA (Reference l, hereby incorporated by reference). All 4ynthetic oligonucleotides were made by the phosphoramidite method using an Applied BioSystems 380A synthesizer.
The qtrategy used is described in Figure 3. The ~AC
promoter was introduced into pGEM-1 as a HindIII-Bam~I fragment to yield pGEM-TAC (steps 1-3). The oncomodulin coding ~equence wa~
obtained from pONCO-4 (~eference l, hereby incorporated by reference), after digestion with HindIII followed by treatment with ~al31 (which deleted the 5'-non coding sequence and part of the coding region), and finally digestion with Dra I (steps 5-6).
The blunt ended fragmentq were cloned into pGEM-TAC, which had been prepared by dige~tion with 8amHI and the resulting ends filled with the large fragment of DNA polymerase I (Klenow) (step 4). A subclone in which a BamHI restriction site had been re-generated was chosen (step 7), and the junction sequenced. This plasmid was then linearized with BamHI and the ends were rendered blunt by treatment with mung bean nuclease (step ~). A double stranded DNA fragment of 15 base pairs (step 9), composed of two complementary ~ynthetic oligonucleotides, was then introduced between the TAC promoter and the oncomodulin sequence in order to restore the entire coding region (step l0). In addition, this generated a new unique Cla l restriction site and a Shine/Dalgarno ~equence, AGGA, 4 nucleotides upqtream of the start codon.
The construction was designed so that the Shine-Dalgarno polymerase binding sequences were immediately upstream of the ATG
start codon of the oncomodulin coding sequence. A new, unique r~ ;f 13 Cla 1 restriction site was also created by this ligation, which proved useful in the screening ~or the desired recombinant. The re~ulting nucleotide sequence of the junction between the TAC
promoter and the oncomodulin coding ~equence in the new plasmid is SEQ ID NO:2~, shown in Figure 4. The sequence was determined from the SP6 promoter region, which is upstream of the TAC promoter, by the dideoxy chain termination method (Reference 3, hereby in-corporated by reference).
Oncomodulin Expression and Purification: The plasmid pGEM-TAC-ONCO was uRed ~uccessfully to transform E. coli JM101, JM103, DH5, or GW5889, the latter being lon- (i.e. free of endo-genous lon protease). The transformants were screened for oncomo-dulin production. The isolation of oncomodulin from bacteria was ba~ed on procedures used for calmodulin (Reference3 4, 5, hereby incorporated by reference). Bacteria were harvested from 2L of culture in L-broth by centrifugation, and the pellet (approx. 5-7 g) requspended in 20 mL of 2.4 M sucrose, 40 mM Tris-HCl, 10 mM
EDTA, pH 8Ø The suspension was incubated on ice for 30 min.
80mL of 50 mM Hepes-HCl, 100 mM KCl, 1 mM E~TA, 1 mM dithio-threitol, 100 ~g/ml lysozyme were then added, and the bacteria lysed at 4C overnight. The lysed bacteria were centrifuged a~
40,000g for 30 min in a Beckman LS65 ultracentrifuge (60Ti rotor).
The clear supernatant was heated rapidly in a boiling water bath, held between 65-80C for 5 min, and cooled immediately on ice.
The denatured proteins were removed by centrifugation at 40,000g for 30 min. The oncomodulin in the resulting supernatant was isolated by ammonium sulph~te precipitation, followed by sequen-tial ion exchange and gel filtration as previously described ~or extracts of tumour tissue (References 6, 71 hereby incorporated by reference).
Oncomodulin Mutagenesis: The HindIII to XmaI fragment of pGEM-TAC-ONCO (Figure 3) was subcloned into the polylinker region of pTZ19R (Reference 8, hereby incorporated by reference;
~Pharmacia PL Biochemicals) to produce pTZl9-Onco. Single strand-ed DNA was obtained from the resulting recombinant after infection of a bacterial culture with helper phage M13K07 (Pharmacia PL) using the procedure of Zoller and Smith (Reference 9, hereby in corporated by reference). A synthetic oligonucleotide (21mer) containing the desired mutation (eg for Glu 59:
TGGATACCTCGAGGGAGATGAG (SEQ ID NO:26)) was used to prime the syn-the~is of the second DNA strand before transformation of competent E. coli JM103. A new XhoI site was al80 created which was useful in screening. The kinase labeled oligonucleotide was also used to screen the resulting transformants, and the nucleotide sequence of the choice candidates was determined by the dideoxy chain termina-tion method (Reference 3, hereby incorporated by reference) (Figure 5).
Characterization of ~ecombinant Oncomodulin: Ultra-violet spectra were obtained with a Beckman DU8 spectrophotometer using oncomodulin at 5 mg/mL in a buffer consisting of 10 mM
sodium cacodylate, 150 mM KCl, 1 mM dithiothreitol pH 7.0, which had been pas~ed over the cation exchange resin Chelex lOQ (BioRad Laboratories). The resulting working buffer had a residual cal-cium concentration, estimated by atomic absorption spectrometry , 7 ~ ~

(Pye Unicam SPl91), of less than 0.002 mM.
The fluorescence spectra were obtained with SLM 8000C
spectrofluorimeter equipped with Neslab Endocal ~TE-5DD circuLat-ing bath. The spectra were corrected for contributions of the blank, and normalized for comparison. The fluorescence (~ ex = 280 nm~ of a solution of oncomodulin in the cacodylate buffer described above, was measured in 0.5 cm quartz cuvettes at 20C (a~sorbance reading~ at 280 nm approximately 0.05). The excitation and emission bandpasses were both 4 nm.
The W spectra of bacterially expressed oncomodulin and rat hepatoma oncomodulin are shown in Figure 6. Antigenic compar-ison of the recombinant oncomodulin with the native protein from hepatoma was made using an immunoradiometric assay (Reference 11, hereby incorporated by reference). The re~ults are illustrated in Figure 8.
The tryptic peptide~ of recombinant oncomodulin were separated by reverqe-phase HPLC, and their amino acid composition and sequence obtained as described (Reference 12, hereby incorpor-ated by reference).
Con~truction of pGEM-TAC-ONCO-CI
The plasmid pGEM-TAC-ONCO which contain~ the DNA ~e-quence encoding the rat oncomodulin protein, under the control o~
the bacterial TAC promoter, described above, was digested with the restriction enzymes EcoRV and S~tI, each cutting a unique 3ite in the DNA. The two fragments thus obtained were separated by gel electrophoresis and the large fragment was recovered by electro-elution. Two complementary oligonucleotides whose sequence (SEQ

C,t~ '7 ~ ~

ID N0:29) is shown in Figure 9 were synthesized on an Applied BioSystem DNA synthesizer model 380A. (Nucleotide triplets can of course be replaced by other triplets coding for the same amino acid.) Equimolar amounts of the two oligonucleotides were mixed in a colution 10 mM TRIS pH 8.0, 1 mM EDTA and 0.2 M NaCl at room temperature for 20 minutes. The annealed oligonucleotides were then ligated to the large EcoRV-S-~tI fragment of pGEM-TAC-O~C0 thus restoring the reading frame of the oncomodulin ~equence.
This new pla~mid DNA was used to transform DHS cells from which the modified oncomodulin protein is extracted. The general methods used for growing the bacteria, purifying the plaqmid DNA, restriction enzyme digestion, ligation of DNA fragments and trans-formation of the bacteria are those described in several molecular biology technique books such as "Molecular Cloning" by T. Maniatis, E.F. Fritsch and J. Sambrook, Cold Spring Harbor Laboratory, 1982.
The nucleotide ~equence of the recombinant plasmid DNA
molecule was checked by DNA sequencing through the modified region by the chain termination method of Sanger using oligonucleotide primers flanking both 3ides of this region.
Expres3ion and Purification of Recombinant Proteins The methods used to extract from bacteria and to purify the mutated recombinant oncomodulin are those de~cribed above in relation to the extraction and purification of native recombinant oncomodulin.
Titration with Terbium Example II: Native oncomodulin:
Protein was dissolved in 10 mM PIPES, 100 mM KC1, pH 6.5 ,77~
to a final concentration of 45 M. The protein sample was excited at 285 nm in an SLM 8000 C fluorimeter, and the emission at 545 nm was monitored. The emission and excitation band pass were 4 nm and the temperature was 20C. This ample, which gave an emission at 545 nm due to the fluorescence of Terbium, was titrated with increasing additions of the metal to the protein solution. The emission was measured after each of 45 2 ml aliquots of 2.5 mM
TbC13 in PIPES buffer. The resulting fluorescence was plotted against the ratio of Terbium to protein concentration (Figure 10).
The bacterially expressed native oncomodulin had an increased Terbium luminescence with increasing Terbium addition, which reached a maximum at approximately 2Tb/mole of protein.
Thi~ was not unexpected because of the presence of 2 binding ~ites in the oncomodulin ~tructure shown by X-ray crystallography, and the ability of oncomodulin to bind only 2Catmole (Reference 13, hereby incorporated by reference). It was also in agreement with published results on Tb binding to oncomodulin purified from rat liver tumours (Reference 14, hereby incorporated by reference).
The increase in fluorescence was concluded to be due to energy tran~fer from Tyr~57 and/or Tyr 65, which are the only two tyro-sines in the native molecule. About one third of the total Terbium fluorescence was produced when 0-1 Tb/mole was bound, with the major fluorescence output occurring when 1-2 Tb/mole of protein were bound. This was interpreted to mean that the EF ~ite (the ~ite with the highe~t affinity) was the one filled first but ~ ~:3 ~3 ~
with the minor contribution to overall fluore~cence, while Tb binding to the CD ~ite led to the major emission signal.
The replacement of Tyr 65 with Phe 65 caused an unexpec-ted 30% increase in the Terbium luminescence fluorescence (Figures 10 and 11~. While not wishing to be bound by any theory, it is suggested that the substitution of Tyr 65 by Phe 65 may have re ulted in a conformational change in the protein which results in enhanced energy transfer from Tyr 57-to bound Terbium.
The replacement of Asp 59 with the longer Glu 59 caused a doubling in the Tb emission (Figure~ 10 and 11). While not wishing to be bound by any theory, it i9 suggested that the Glu 59 eliminated a water bridge between Asp 59 and the chelated Tb and this elimination of water reduces the quenching by solvent, which subsequently leads to increased Terbium fluorescence. Also a conformational change that brings the Tb slightly closer to the Tyr 57 energy donor cannot be ruled out.
The substitution of Tyr 65 by Trp 65, or Phe 102 by Trp 102 was designed to increase Tb emission by providing a better energy transfer donor. Thus the decrease in Tb signal from both Trp 65 and Trp 102 mutants of oncodulin was unexpected. It i9 quggested that, the presence of tryptophan 102 or tryptophan 65 provides an alternative competitor for energy transfer from Tyr 57 to the bound Terbium.
The~e observations on single amino acid substitution result in the conclusion that Tyr 57 i~ an important source of energy for excitation of Terbium in both the CD and EF sites. It is evident that the extent of Tb emission can be modified by 2~ 7~
protein engineering.
Example III: Construct I:
Protein was dissoLved as in Example II to a final con-centration of 8 ~M. The titration with Terbium was performed as in Example II.
The titration of Construct I, where 10 amino acids in a linear sequence in oncomodulin were changed so that the modified oncomodulin molecule included a chelator ~equence of the inven-tion, caused a dramatic improvement in the emission signal from Tb, which was nearly 20 fold better than that of native oncomodu-lin (Figures 10 and 11). Also noticeable was that the major fluorescence change now occurred when 0-1 Tb/mole protein was bound. This contrasts with native oncomodulin, where the major fluorescence change occurs when between 1-2 moles of Terbium per mole of oncomodulin are bound.
Example IV:
This example demonstrates Resonance Energy Transfer (RET) from aromatic amino acid~ (tyrosine and tryptophan) to bound Terbium in oncomodulin and mutant proteins.
The experiments were performed as in Example~ II and III, except that t~he fluorescence from the aromatic amino acid rather than luminescence from Terbium wa~ measured during the titration. In the ca~es where tyrosine wa~ the only aromatic amino acid, (Glu 59 mutant protein and native protein), the protein fluroescence (originating from tyrosinej was monitored at 310 nm (Figure 12). In the ca~e where tryptophan was also one of the aromatic amino acid residues in the protein, (Construct I and 2'7 ~ ~3 Trp 102), the protein fluorescence due to tryptophan was monitored at 350 nm (Figure 13).
The fluorescence intensity in each of these caseY
decreased on addition of Terbium. The data plotted in Eigures 12 and 13 represent~ the fractional change in fluoreQcence Fo~
Fn/Fo versus ~Tb3+]/~Protein], where Fo is the fluorescence in~ensity in the absence of Terbium and Fn is the fluorescence intenqity after the addition of Terbium~.
In all ca3es, the fractional fluorescence change reached a plateau value at a ~Tb3+]/[Protein] ratio of 2:1. The fraction-al fluorescence change wa~ greater for Glu 59 than for native, paralleling the increased Terbium luminescence seen in Figure 10.
This showR that RET from the tyrosine of the Glu 59 mutant to bound Terbium was more efficient than that of native protein. In the case of Construct I, E'igure 13 clearly shows that tryptophan located in pGsition 57 has a high R~T efficiency especially when compared to that of the Trp 102 mutant of oncomodulin. This parallels the increased Terbium luminescence seen in Figure 11 of Construct I. This ~hows that efficient RET from the tryptophan to Terbium re~uires that the two species be in close proximity to one another.
Exam~le V:
This example demonstrates the use of Construct I protein to assay Terbium in low concentrations.
Stock solutions of Terbium in 10 mM PIPES, lO0 mM KCl, pH 6.5 buffer were diluted to the concentration range shown in Figure 14. Ten microlitres of a stoc~ solution of 0.57 mg/ml of ~;~`s~
Construct,I protein in the qame buffer was added to 1.5 ml samples of each of the Terbium solutions.
The luminescence at 545 nm of these solutions was measured as outlined in Example II, except that the emission band-pass was 8 nm. The luminescent signal of a blank solution con-taining no Terbium was measured under identical conditions and subtracted from the signal of the samples. Figure 14 shows that the response of the lumine~cent signal for Terbium is linear over nearly 3 orders of magnitude of Terbium concentration. A sample with a Terbium concentration as low as 7X10-13 M could be detec-ted. This corresponds to a sample of 1.5 ml which contains only 1.05x10-15 moles of Terbium tor 6X108 ions).
ExamPle VI:
This example demonstrates the use of a Terbium binding assay to replace a qualitative radioac~ive 45Ca assay which can be used to detect the presenc0 of a protein containing a chelator sequence.
oncomodulin mutant Trp 57 was purified ~rom bacteria as outlined above (cf, Oncomodulin Expres3ion and Purification).
Figure 15 shows the final stage of purification by gel filtration on Sephadex G50. ~ach fraction was monitored for the presence of protein by measurement of absorbance at 280 nm. Aliquotq from each fraction were assayed for calcium-binding properties with a Chelex assay (Referenceq 6 and 7, hereby incorporated by referen~
ce) employing radioactive 45Ca and a scintillation counter.
Aliquot~ were also monitored for the presence of a chelator sequence that show~ enhanced Terbium luminescence upon 2.~27~
binding Terbium. 20 ~L aliquots were diluted in 1.25 ml of a 10 ~M PIPES, 190 mM KCl buffer at pH 6.5. 5 ~L of a ~olution of 5 mM TbC13 solution was added to each aliquot, which led to the formation of luminescent complexes when the protein was present.
The resulting fluorescence intensity was plotted in Figure 15 along with the values from the absorbance and the radioactive calcium assay.
Figure 15 demonstrate~ that the addition of Terbium to a protein capable of forming luminescent complexec can be used for the detection of such proteins. Further, this assay avoidQ the hazards of handling radioactive material~, and the cost of radio-active disposal. The number of manipulative teps i8 also much reduced, ~aking the assay simpler and quicker to perform.
Example VII:
A 14mer, with cysteine at position 7, of the following ~tructure wa~ prepared SEQ ID NO:20:

Gly A~p Lys Asn Ala Asp Gly Cys Ile Glu Phe Glu Glu Leu The chromophore ~-(l-pyrenyl)iodoacetamide (PIA) was reacted with the 14mer at about pH8 in a ~Cl/Tri~ buffer and thereby covalently bonded to the cysteine at position 7 of the 14mer. Samples of 10 ~M PIA-14mer in lOmM PIPES, pH 7.2, (200 ~L) were titrated with 5 ~L aliquots of stock solutions of Tb3+ or Eu3+ of concentrations 100 ~M and lmM. Fluorescence of the chelated lanthanides was measured and plotted in Figure 16A (lOO~M
concentration) and Figure 16B (lmM) concentration. Results for Tb3+ are plotted as ~quares and for Eu3+ are plotted as triangles.

?!~8277~
The data were not dilution corrected.
Example VIII:
500 ~L aliquots of 10 ~M ~olutions of the PIA-14mer of Example VII were reacted with 5 ~L additions of lmM Tb3+, lmM Eu3+
and 3mM Ca2+, respectively, in 10 mM PIPES at pH 7.2. The effect of light scattering on the chelated metal ions was observed, with excitation at 344 nm and emission at 688 nm. Results are given in Figure 17, trianglec indicating Tb3+ circles indicating Eu3+ and squares indicating Ca2+. Data were corrected for instrument response and dilution.
Example IX:
The 14mer of Example VII wa~ covalently bonded via cysteine at the 7-position to the chromophore 7-diethylamino-3-((4'-iodoacetylamino)phenyl)-4-methylcoumarin (DCIA). 10 ~M
aliquots of the DCIA-14mer in lOmM PIPES pH 7.2 (200 ~L) were titrated with Eu3+ at concentrations of 100 ~M and lmM. Lumines-cence was measured and result-~ are given in Figures 18A (100 ~M) and 18B (lmM).
Example X;
A 23mer, with cysteine at position 7, of the structure identified above as SEQ ID N0:21 was covalently bonded at the 7-position to DCIA. Stock solution of Eu3+ (100 ~M) was added to a 2 ~M DCIA-23mer sample in 200 ~L of 50mM PIPES, pH 7Ø Stock solution of Eu3+ was al~o reacted with the DCIA-14mer under the same conditions. Luminescence was measured and re~ults are given in Figure 19 (DCIA-14mer, open triangles; DCIA-23mer, filled tri-an~les).

2~'~27~/ ~

Example X_:
Oncomodulin was modified, by cassette mutagenesis, to replace the naturally occurring CD loop by the sequence identified above as SEO ID ~0:22 and the natusally occuring cy~teine at position 18 of oncomodulin was removed by site specific mutagenesis and replaced by valine.
(Construct 3) The DCIA chromophore was covalently bonded to the cysteine mGiety at the 7-position and subsequently reacted with Eu3~ at variou~ concentrations in 150mM KCl, lOmM PIPES, pH 7Ø
Luminescence was measured and results are given in Figure 20.
Eu3+ (10 ~M stock) was added to DCIA-Construct 3 (1 ~M) in 200 ~L of 150mM RCl, lOmM PIPES, pH 7Ø Luminescence was mea-~ured and results are given in Figure 21.
Example XII:
The chromophore iodoacetamidosalicylic acid tIASA) was covalently bonded to the cysteine iety at position 7 of Construct 3. Tb3~ tlO ~M stock) in different qualities was added to the IASA-Construct 3(1 ~M) in 200 ~L of 150mM KCl, 10mM PIPES, pH 7Ø Lumiscence was measured and results are given in Figure 22, graphed again~t the quantity of Tb3+ used. In a separate experiment, the quantity of Tb3~ was maintained at 2 ~M and reacted with varying amounts of IASA-Construct 3 in 200 ~L of 150mM RCl, lOmM PIPES, pH 7Ø Results are given in Figure 23.
Exam~le XIII:
Chromophores were attached to the cysteine in Construct 3 and evaluated for Tb3+ and Eu3~ sensitization.

~ ~ 2 7 r~ ~

Modified Construct 3 (1.5 M) _ Eu3~ Counts ?b3+ Counts PIA 220,000 350,000 PMIA 160,000 386,000 DIMS 230,000 745,000 BPIA 32,000 280,000 ~Ln3+~ = 5~M.
Abbreviation~;
PIA - N-(l-pyrenyl)iodoacetamide.
PMIA - l-pyrenemethyl iodoacetate.
DIMS - 4,4'-dimaleimidyl~tilbene.
BPIA - benzophenone-4-iodoacetamide.

;J ~ ~ f~

REFERENCES
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8. Mead, D.A., Szczesna-Skorupa, E. & Remper, B. (1986) Protein Engineering 1, 67-74.

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Biophyq. Acta 872, 16-23.

15. Houghten, R.A. (1985) P~S 82 5131-5135 16~ Merrifield, R.B. (1963) J. Amer. Chem. Soc. 85 2148-2154 17. Houghten, R.A., DeGraw, S.T., Bray, M.K., Hoffman, S .R. and Frizzel, N. D. (1985) Int. J. Pept. Prot. Res. 27 673-678 2 ~ 1 rl ~
SEQUENCE LISTING

(1) GENEXAL INFORMATION:
~i~ APPLICANT: Banville, Dennis Macmanus, John P
Marsden, Brian Szabo, Arthur G
Hogue, Christopher Sikorska, Marianna Clark, Ian (ii) TITLE OF INVENTION: Engineered protein chelates suit~ble for fluorescent lanthanide (e.g. terbium (III)) based tim~
resolved fluorescence assays ~iii) NU~BER OF SEQUENCES: 30 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Smart & Biggar (B) STREET: P.O. Box 2999, Station D
(C) CITY: Ottawa ~D) STATE: Ontario (E) COUNTRY: Canada (F) ZIP: KlP 5Y6 (v) COMPUTER READABLE PORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
tA) APPLICATION NUMBER: US 07/476757 (B) FILING DATE: 01-APR-1990 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Wheeler, Michael E
(C) REFERENCE/DOCRET NUMBER: 63247-223 CIP
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613)232-2486 (B) TELEFAX: (613)232-8440 (C) TELEX: 053-3731 2 ~ 7 1 ~3 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 12 amino acids tB) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECVLE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Asx Xaa Asx Xaa Asx Xaa Xaa Xaa Glx Xaa Glx Glx , 10 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Asx Xaa Asx Xaa Asx Xaa Xaa Xaa Glx Xaa Xaa Glx (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Asp Lys Asn Ala Asp Gly Trp Ile Glu Phe Glu Glu (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 2 ~5~C2 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Asp Lys Asn Ala Asp Trp Gly Ile Glu Phe Glu Glu (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRlPTION: SEO ID NO:S:
Asp Lys Asn Ala Asp Ala Trp Ile Glu Phe Glu Glu (2) INFORMATION POR SEQ ID NO:6:
(i) sEguENcE CHARACTERISTIC5:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Asp Lys Asn Ala Asp Gly Trp Ile Glu Trp Glu Glu (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

~ ~J ~ 7 7 ~

Asp Lys Asn Ala Asp Ala Trp Ile Glu Trp Glu Glu (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Asp Lys Asn Ala Asp Trp Gly Ile Glu Trp Glu Glu (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) 5EQUENCE DESCRIPTION: SEQ ID NO:9:
Gly Asp Lys Asn Gly Asp Gly Trp Ile Glu Phe Glu Glu Leu (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYP~: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Gly Asp Lys Asn Gly Asp Gly Tyr Ile Glu Phe Glu Glu Leu '7 ~

(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCB DESCRIPTION: SEQ ID NO:ll:
Gly Asp Lys Asn Gly Asp Gly Phe Ile Glu Tyr Glu Clu Leu (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lin~ar (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Gly Asp Lys Asn Gly Asp Gly Tyr Ile Glu Trp Glu Glu Leu (2) INFORMATION FOR SEQ ID NO:13:
(i) SEOUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: 1 inear (ii) MOLECULE~TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEO ID NO:13:
Gly Asp Asn Asp Gln Ser Gly Tyr Leu Asp Gly Asp Glu Leu (2) INFORMATION FOR SEO ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids ?J ~ ~ ~ r~

(~) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptid~

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Gly Asp Asn Asp Gln Ser Gly Trp Leu Asp Gly Asp Glu Leu (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 12 amino acids ~B) TYPE: amino acid (D) TOPOLOGY: linear lii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Asp Xaa Asn Xaa Asp Xaa Xaa Xaa Glu Xaa Glu Glu (2) INFORMATION FOR SEQ ID NO:16:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (3) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TS'PE: peptide (xi) 5EQUENCE DESCRIPTION: SEQ ID NO: 16:
Asp Xaa Asn Xaa Asp Xaa Trp Xaa Glu Xaa Glu Glu (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: 1 inear (ii) MOLECULE TYPE: peptide 2 ~ 7 7 ~

rxi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Asx Xaa Asx Xaa Asx Xaa Xaa Xaa Glx Xaa Xaa Glx (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Asx Xaa Asx Xaa Asx Xaa Xaa Xaa Glx Xaa Glx Glx (2) INFORMATION FOR SEQ ID NO:l9:
(i3 SEOUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids ~B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:
Asp Xaa Asn Xaa Asp Xaa Trp Xaa Glu Xaa Xaa Glu (2) INFORMATION FOR SEO ID NO:20:
(i) SEOUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid tD) TOPOLOGY: llnear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Gly Asp Lys Asn Ala Asp Gly Cys Ile Glu Phe Glu Glu Leu ~J ~i ~ r~

(2) INFO~MATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Ser Leu Val Ala Leu Asp Asn Asn Ala Asp Gly Cys Ile Glu Phe -5 1 5 l0 Glu Glu Leu Ala Thr Leu Val Ser (2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Asp Lys Asn Ala Asp Gly Cys Ile Glu Phe Glu Glu (2) INFORMATION FOR SEQ ID NO:23:
(i) 5EQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS- single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CACACAGGAA ACAGGATCGA TGAGCATCAC GGACATC

2~3~277~

(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH~ 57 base pairs (B) TYPE: nucleic acid (C) STRAN~EDNESS: double (D~ TOPOLOGY: linear ~ MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GATATCTTCC GGTTCATAGA CAACGACCAG AGTGGATACC TGGATGGAGA TGAGCTC

~2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
GATATCTTCC GGTTCATCGA TAAGAACGCG GATGGATGGA TAGAATTCGA GGAGCTC

(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
TGGATACCTC GAGGGAGATG AG

Claims (23)

1. A chelator sequence selected from the group consisting of sequences of twelve amino acids containing the following amino acids and sequences of the said amino acids in which one or more of the said amino acids is bonded to a chromophore.

2. A sequence according to claim 1 containing the following amino acids

3. A sequence according to claim 1 selected from the following sequences of amino acids

4. A chelator sequence according to claim 1 having at one of positions 1, 3, 5, 7, 9 or 12 of the twelve amino acid sequence an amino acid covalently bonded to a chromophore.

5. A chelator sequence according to claim 1 having, at position 7 of the twelve amino acid sequence, cysteine which is covalently bonded to a chromophore selected from the group consisting of N-(1-pyrenyl)iodoacetamide, 1-pyrenemethyliodoacet-amide, 4,4'-dimaleimidylstilbene, benzophenone-4-iodoacetamide, iodoacetamidosalicylic acid and 7-diethylamino-3-((4'-iodoacetyl-amino)-phenyl)-4-methylcoumarin.

6. A sequence according to claim 1 which sequence replaces amino acids between the amino acids 50 and 63, 89 and 102 or 15 and 28 of native oncomodulin.

7. A sequence according to claim 1 which sequence is sequence SEQ ID NO:3 and which replaces the amino acids between acids 50 and 63 of native oncomodulin.

8. A method of chelating a metal which comprises reacting the metal with a chelator sequence of amino acids as claimed in claim 1 under conditions in which the metal and sequence of amino acids react to form a chelate.

9. A method according to claim 8 wherein the metal is a luminescent lanthanide.

10. A method according to claim 8 wherein Terbium or Europium is reacted with oncomodulin which has been modified by replacement of the amino acids between acids 50 and 63 of native oncomoduLin with sequence SEQ ID NO:3 under conditions in which the luminescent lanthanide and the sequence of amino acids react to form a chelate.

11. A method of chelating a metal which comprises passing a metal-containing vehicle over a chelator sequence of 12 amino acids as claimed in claim 1, which sequence is immobilised on a solid phase.

12. A complex comprising a luminescent lanthanide and a chelator sequence selected from the group consisting of sequences of twelve amino acids containing the following sequence:

and sequences of the said amino acids in which one or more of the said amino acids is bonded to a chromophore.

13. A complex according to claim 12 wherein the chelator sequence of twelve amino acids contains the following acids:

wherein Xaa in the 7 position is Trp, Tyr or Phe or a derivative of Trp, Tyr or Phe, or Cys bonded to a chromophore.

14. A complex according to claim 12 wherein the chelator sequence of twelve amino acids contains the following acids:

wherein Xaa in the 2, 4, 6, 8, 10 or 11 position is Trp, Tyr or Phe or a derivative of Trp, Tyr or Phe, or Cys bonded to a chromo-phore.

15. A complex as claimed in claim 12 wherein the chelator sequence is selected from the sequences SEQ ID NOS: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 22, 20 and 21.

16. A complex as claimed in claim 12 wherein the luminescent lanthanide is Terbium, Europium or Samarium.

17. A complex according to claim 12 wherein the chelator sequence has, at position 7 of the twelve amino acid sequence, cysteine which is covalently bonded to a chromophore selected from the group consisting of N-(1-pyrenyl)iodoacetamide, 1-pyrenemethyl iodoacetamide, 4,4'-dimaleimidylstilbene, benzophenone-4-iodoacet-amide, iodoacetamidosalicylic acid and 7-diethylamino-3-((4'-iodo-acetylamino)-phenyl)-4-methylcoumarin.

18. A complex as claimed in claim 12 wherein the chelating sequence replaces amino acids between the amino acids 50 and 63, 89 and 102 or 15 and 28 of native oncomodulin.

19. A complex as claimed in claim 12 wherein the amino acids between acids 50 and 63 of native oncomodulin have been replaced by the sequence SEQ ID NO:3.

20. A method of assay which comprises forming a fluorescent complex as claimed in claim 12 and observing the fluorescence of the complex.

21. A method of assay which comprises forming a complex of a luminescent lanthanide chelated to a sequence comprising 12 amino acids wherein positions 1, 3, 5, 7, 9 and 12 of the sequence are occupied by amino acids that have side chains containing atoms that can donate lone pairs of electrons to the chelated lanthanide and the acid at position 7 contains an aromatic ring.

22. A method according to claim 21 wherein in the twelve amino acid sequence the acid at position 7 is tryptophan.

23. A kit for simultaneous detection of two analytes which kit contains Terbium in a complex as claimed in claim 12 and also contains Europium bound to an organic non-proteinaceous chelating agent.