WO1985005638A1

WO1985005638A1 - Enzyme hydrazides

Info

Publication number: WO1985005638A1
Application number: PCT/US1985/000992
Authority: WO
Inventors: Meir Wilchek; Edward A. Bayer; Jonathan M. Gershoni
Original assignee: Meir Wilchek; Bayer Edward A; Gershoni Jonathan M
Priority date: 1984-05-29
Filing date: 1985-05-29
Publication date: 1985-12-19
Also published as: IL71947A0; EP0186692A4; IL71947A; EP0186692A1

Abstract

A hydrazide derivative of an enzyme wherein the hydrazine or hydrazide moiety is linked to the enzyme molecule either directly or via a functionally inert spacer, via a functionally active molecule or via an antibody directed to the specific enzyme. The hydrazine or polymeric form of hydrazide is preferably directly covalently linked to the enzyme, which linkage is advantageously via a functional group not effecting enzymatic activity selected from amino, carboxyl, aldehydo or sulfhydryl groups of the enzyme. There is further provided a method for the detection and determination of glycoconjugates for gel electrophoretic analysis of such glycoconjugates and the blots thereof, for the microscopic localization of such glycoconjugates, for the detection of macromolecules containing amino or aldehyde groups, carboxy or vicinal hydroxy groups, for the screening of monoclonal antibodies, which comprises interacting same with a hydrazido-enzyme conjugate according to claim 1, and utilizing the enzyme thereof for visualization or other detection and determination.

Description

ENZYME HYDRAZIDES FIELD OF THE INVENTION:

There are provided hydrazido derivatives of enzymes (enzyme hydrazides) for the selective staining or laoeling of various macromolecular species. Procedures involving the use of such enzyme hyαrazides may be rendereα specific chemically for proteins, glycoconjugates, sialoglycoconjugates, galactoglycoconjugates, nucleic acids, as well as naturally existing or chemically introduced carDonydrate-containing, aldehyde-containing, amino-containing, and carboxyl-containing macromolecules. The latter target compounds may be attached to solid supports, e.g. membrane filters, microtiter plates or intact cells, and the described procedures may be used for the detection, localization and quantification of the desired target compound.

BACKGROUND OF THE INVENTION:

The αetection, localization, identification ana assay of biologically active macromolecules are achieveα by a variety of analytical methods. The latter are usually accomplished by a comoination of physical-chemical separation together witn selective staining or labeling procedures. For example, a complex mixture of proteins can be separated according to size by polyacrylamide gel electrophoresis in the presence of sodium dodecyl suifate (SUS-PAGE) or according to charge by isoiectric focusing (IEF), and the pattern of the individual protein bands can oe visualized oy a general stain for proteins, e.g. Coomassie Brilliant Blue or Amido Black. Alernatively radiolaoled proteins may be visualized on gels by autoradiograpny.

Nucleic acids, resolved in agarose or in polyacrylamide gels, can be detected with Coomassie Brilliant Blue or via interaction with the fluorescent dye ethidium bromide. Sugar-containing proteins (glycoproteins) and other sugar-containing macromolecules, e.g. glycolipids, mucopolysaccharides, lipopoly-saccharides, peptidoglycans, and simple homo- or heteropolysaccharides, can also be visualized by various techniques.

The phenomenal variety of biologically active carbohydrates is generated by the natural abundance of monosaccharide types, the range of possible glycosidic linkages between two sugars, and the two anomeric configurations. In contrast to this biological diversity, chemically the different types of sugars are very similar, since the hydroxyl group represents the major accessible chemcially reactive moiety. The availablility of chemical reactions which would be selective for labeling saccharide-containing macromolecules is therefore severely limited. In practice, only one enzymatic reaction

(i.e. the specific oxidation of D-galactose and

N-acetyl-D-galactosamine by the enzyme galactose oxidase, E.C. 1.1.3.9) and only one mild chemical reaction (i.e., periodate oxidation of vicinal hydroxyl groups) are currently in use to any great extent, both of which generate aldehyde groups on sugars. The resultant aldehydes can then be labeled specifically by chemical means.

One of the most prevalent techniαues for labeling oxidized sugars has been the formation of Schiff's bases with fuchsin (F.H. Kasten (1960) Int. Rev. Cytol. 10:1-100). Alternatively, the oxidized sugars can be labeled by radioactive reagents (e.g., tritiated sodium borohydride) and the latter can be visualized by autoradiography or radiofluorography.

Whereas in the past, characterization of resolved polypeptides in gels has been demonstrated (e.g. general staining of proteins with dyes such as Coomassie Brilliant Blue or of glycoproteins by periodic acid-Schiff stain (PAS), specific detection of glycoconjugates via lectins or of antigens by antibodies, etc.), such methodologies are cumbersome and quite often inefficient. However, analysis of constituents of electrophoretograms has become more effective by the recent introduction of new and improved methooolgy by which macromolecules are first eluted from the gel and immobilized onto a suitable matrix prior to staining, a process termed "blotting" (J.M. Gershoni and G.E. Palade (1983) Anal. Biochem. 131:1-15). This procedure, has many inherent advantages, including (a) increased facility in handling, (b) improved accessibility of immobilized macromolecules to various reagents, (c) reduction in sample size, (d) reduction in processing time, (e) possibility for producing multiple replicas of single gels, (f) transferred patterns can be stored for long periods of time, (g) a single pattern can be subjected to multiple successive analyses, and (h) blots are amenable to analytical procedures impracticable on gels.

Due to the experimental facility afforded oy clotting procedures, much effort has been directed toward designing improved methods for general ano selective staining (or labeling) procedures applicable to this techniαue. Although general staining of proteins immobilized (biotted) onto nitrocellulose has been reported, the general staining of glycoconjugates has yet to be described.

SUMMARY OF THE INVENTION:

There is provided a novel type of staining or labeling process which is oased on the chemical interaction of enzyme hydrazides with a variety of target macromolecules. This process combines the chemical specificity and stability of the hydrazide moiety with the sensitivity and amplificatory properties of enzymes previously used in various staining procedures and assays. The process can be used to stain aldehyde-containing, amino-containing or carboxyl-containing macromolecules and is suitable for blotting techniques, gels, solid-phase assay systems as well as for light and electron microscopic cytochemistry. 1. Preparation of enzyme hydrazides:

Enzyme hydrazides can be prepared in numerous ways. Several representative examples will oe described herein. Essentially, functional hydrazide residues or polymeric forms of hydrazides can be linked to a functional enzyme either (a) directly by chemical (covalent) means, (b) bridged via a functionally inert spacer, or (c) bridged via an intermediate functional molecules(s), e.g. via the avidin-biotin complex or via an antibody directed toward the enzyme. 1.1 Simple direct hydrazidation of endogenous enzyme carboxyls:

Hydrazides can be covalently linked to glutamate, aspartate and

C-terminal carboxyl residues of enzymes via interaction with hydrazine

(Ed. 1a) or dihyorazides (e.g. maleic, succinic or adipic dihydrazide;

Ed. 1b) in the presence of a water soluble carbodiimiαe (WSC), e.g. l-ethyl-3-(3-dimethylamino-propyl) carbodilmide.

(1.1a) ENZ-COOH + NH₂NH₂ ------------ ENZ-CONHNH2

WSC

(1.1b) ENZ-COOH + R-(CONHNH₂)2 --------ENZ CONHNHCO-R-CONHNH₂

WSC S 1.2 Simple direct hydrazidation via enzyme amino groups:

The enzyme can be reacted successively with an excess of dialdehyde (e.g. glutaraldehyde) followed by an excess of dihydrazide (i.e. maleic, succinic, adipic, etc.):

(1.2) ENZ-NH₂ + R'-(CHO)₂ --------> ENZ-N=CH-R'-CHO

R-(CONHNH₂)₂

ENZ-N=CH-R' -CH=N-NHCO-R-CONHNH₂ Further stabilization of the hydrazone groupsformed can be accomplished by their reduction (e.g. using oorohydride, cyanoborohydride, etc.).

1.3 Simple direct hydrazidation of glycoproteins:

Hydrazide derivatives of glycoprotein enzymes or enzymes which have been glycosylated through chemical or enzymatic (or biological) means, can be prepared by prior activation of the resident sugars by periodate. Alternatively, enzymatic treatment, either by galactose oxidase alone or by combined neuraminidase/ galactose oxidase, will generate aldehydes on appropriate glycoproteins. The activated glycoprotein can then be incubated with a suitable dihydrazide thereby producing the enzyme hydrazide via endogenous carbohydrates.

(1.3) ENZ-(carbohydates) --------------------> ENZ-(carbohydrate)-CHO

10-₄ or R-(CONHNH₂)₂ galactose oxidase

ENZ-(carbohydrate)-CH=N-NH-R-CONHNH₂

Again, the hydrazone group formed can be stabilized by reduction as described in section 1.2. 1.4 Hydrazidation of succinylated (carboxylated) enzyme:

The extent of hydrazidation of a given enzyme can be increased by first succinylating the resident amino groups of the enzyme followed by direct hydrazidation of both the endogenous (y) as well as the succinyl-derived (x) carboxyls on the modified enzyme.

(1.4) (HOOC)_y-ENZ-(NH₂)_x + ₂ O O CO

-----------> (HOOC)_y-ENZ-[NHCO(CH₂)₂-COOH]_x -------------------> (H₂NHNOC)_y-ENZ-(CONHNH₂)_x hydrazidation

1.5 Hydrazioation via enzyme-polyhyorazide conjugate:

A soluole polyhydrazide (e.g. polyacrylic hydrazide, polysialic acio hydrazide, or polyglutamic hyorazide) can oe conjugated(e.g. via glutaraldehyde or WSC) to the enzyme of choice.

(1.5) ENZ + P-(CONHNH₂)_x ---------> ENZ-P-(CONANH₂)_x

WSC or

R'-(CHO)₂

1.6 Hydrazidation of enzyme-conjugated carooxyl-containing polymer:

In order to further increase the amount of hydrazide per enzyme, carboxyl containing polymers (e.g. polyglutamic acid) can also be conjugatedfirstto the enzyme and hydrazidation of the conjugate can then be performed.

(1.6) (HOOC)_y-ENZ-NH₂ + P-(COOH)_x ------> (HOOC)_y-ENZ-NHCO-P-(COOH)_x

lhydrazidation

(H₂NHNOC)y-ENZ-NHCO-P-(CONHNH₂)_x 1.7 Hydrazidation via functional intermediates:

In cases where the enzyme is particularly sensitive to the chemical reactions described above (1.1-1.6), hydrazidation of an enzyme via a functional intermediate may be advantageous. Three examples of this approach are as follows:

Hydrazidation of an antibody which recognizes the enzyme can first oe performed and the antioody-hyαrazioe can be reacted with the target molecule. Suoseouently, the antiocoy-bound target molecule can be incubated with the unmodified enzyme. Alternatively, premade soluble complexes consisting of the enzyme and antibody-hydrazide can oe prepared and these complexes can be interacted with the target molecule.

(1.7a) ENZ + Ab-(CONHNH₂)_x -------> ENZ:Ab-(CONHNH )

Avidin-biotin technology can also serve as an appropriate intermediate. The enzyme can oe oiotinylateα unαer mild conditions which do not significantly affect enzyme activity (E.A. Bayer and M.

Wilchek (1980) Methods Biochem. Anal. 26:1-45). Likewise, avidin-hydrazide can be prepared by one of the above-described methods. The avidin-hydrazide can then oe used to chemically label the target molecule via the hyorazide moiety. Suoseαuent interaction with tne biotinylated enzyme serves to associate the enzyme ana target molecules via the avidin-biotin complex. Similar to antibody mediation, premade hydrazido-avioin/biotinylated enzyme complexes can be used to label the target molecule.

(1.7b) Biotin-ENZ + Avidin-(CONHNH₂)_x--->ENZ-Biotin:Avidin-(CONHNH₂)_x In a similar manner, lectin-hydrazide can oe used to mediate between specific sugar moieties (eitner native or artificially introduced) which reside either on the target molecule and/or on the enzyme probe:

(1.7c) sugar-ENZ + Lectin-(CONHNH ₂)_x------> ENZ-sugar:Lectin-(CONHNH ₂)_x

2. Enzymes that can be hydrazide modified

In principle any enzyme can oe used for the preparation of the new reagent, the only constraint being that the process of adding the hydrazide functional group does not inactivate or interfere significantly with the enzyme activity. As hydrazide moieties may be introduced via many functional groups, e.g. via amino, carooxyl, aldehyoo or sulfhydryl groups, etc., of the protein, one would expect tnat a gentle permissible protocol for each possible enzyme to be modified could be developed.

If the enzyme hydrazide is to oe used for localization purposes via histochemical technioues, the product ootained snoulo be insoluble in aαueous solutions ano of a high contrast color. Numerous protocols of this type have been developed for a plethora of enzymes. References to a few examples are nerewith provided: (2a) Alkaline phosphatase; M.S. Burstone (1962) Enzyme Histochemistry and Its application in the study of Neoplasm. Academic Press,

NY. (2b) Acid phosphatase; M.S. Burstone (1961) J. Histochem. Cytochem.

9:146-153. (2c) 5' Nucleotidase; M. Wacnstein anc E. Meisel (1957) Am. J. Clin.

Pathol. 27:13-23. (2d) Glucose 6-phosphatase; M. Wachstein and E. Meisel (1956) J.

Histochem. Cytochem. 4:592. (2e) Acetylcholine esterase; M.J. Karnovsky and L. Roots (1964) J.

Histochem. Cytochem. 12:219-221. (2f) β-Galactosidase; B.D. Lake (1974) Histochem. 0. 6:211-218. (2g) Dopa oxidase; B.Z. Rappaport (1955) Arch. Pathol. 60:444-450. (2h) Horseradish peroxidase; R.C. Graham and M.J. Karnovsky (1966) J. Histochem. Cytochem. 14:291-302. As is demonstrated from the above examples a variety of enzymes, i.e., phosphatases, oxido-reductases, hydrolases and esterases can be used. This, in addition to the numerous substrates that produce products of a wide spectrum of colors, allows the use of enzyme hydrazides in a multitude of conditions and provide the possibility of double labels which would be of particular importance in cytochemical analyses or diagnostic kits.

Quantitative detection of enzyme hydrazide conjugates is possible by reacting the enzyme with a substrate that renders a soluble product that can be quantified spectrophotometrically.

Thus o-phenyldiamine could be used for detection of horseradish peroxidase or p-nitrophenol phosphate for alkaline phosphatase or o-nitrophenyl galactoside with β-galactosidase, etc.

Another alternative which can provide quantitative detection is to solubilize the precipitated enzyme product in an organic solvent which can then be quantified spectrophotometrically. For example, the fast red product generated in the alkaline phosphatase reaction with napthol- AS-MX phosphate + fast red tr salt is readily solubilized in dichloromethane and can be qunatified at 508nm. 3. Target Macromolecules 3.1 Aldehyde-containing macromolecules

The chemistr involved in the interaction of aldeh des (RCHO) with an enzyme hydrazide (ENZ-CONHNH₂) is as follows:

(3.1a) RCHO + ENZ-CONHNH₂ -------> R-CH=N-NHCO-ENZ

The aldehyde may be endogenous to the target molecule or may be induced chemically prior to subseαuent action with the enzyme hydrazide. For example, amines can be derivatizεd by dialdehydes as described below in formula 2b. In addition, vicinal hydroxyl groups on sugars can be oxidized to aldehydes upon chemical reaction with periodate. The same reaction is relatively specific for sialic acids when the reaction conditions (periodate concentration, temperature) are controlled. Furthermore galactosyl residues can also be selectively oxidized to aldehydes by enzymatic treatment with galactose oxidase. Thus, virtually any procedure by which a given target molecule can be selectively modified to contain aldehyde groups can be employed prior to interaction with an appropriate enzyme hydrazide.

As described in section 1.2, the hydrazone group formed which links the enzyme to the target molecule can be further stabilized by reduction (e.g. borohydride or cyanoborohydride) as follows:

(3.1b) RCH=N-NΗCO-ENZ -------> RCH₂NHNHCO-ENZ

BH₄

3.2 Amino-containing macromolecules

In the presence of a suitable oxidant, (e.g., permanganate) amino-containing macromolecules (R-NH₂) will interact with hydrazides in the following manner:

(3.2a) R-NH₂ + ENZ-CONHNH₂ -------> R-NHCO-ENZ

KMnO₄ An amino-containing macromolecuie can also oe converted to an alαenyde through the action of an excess of dialαehyoe as described in the following formula:

(3.2b) R-NH₂ + OHC-(CH₂)_x-CHO --------> R-N=CH-(CH₂)_x-CHO

Attachment of the enzyme hydrazide is achieved by further treatment as descrioed aoove for aldehydes (section 3.1)

3.3 Carboxyl-containing macromolecules

In the presence of carbodiimide, carboxyl groups will interact with hydrazides as follows:

(3.3) R-COOH + ENZ-CONHNH₂ -----------------> RCONHNHCO-ENZ

WSC Since most of the carboxyl moieties on the enzyme hydrazide have been converted to hydrazides, the carbodiimide would act selectively on the carooxyls of the target compounds. In this case the hyoraziαe functional group on the enzyme is exploited as a nucleophile.

EXPERIMENTAL DESCRIPTION

1. Preparation of Enzyme Hydrazides

The following comprises a representative protocol for hydrazidation of an enzyme. Alkaline phosphatase is given here as a representative example. We nave found that otner enzymes (see list, section 2) can oe modified under conditions in which enzymatic activity is preserved. Binding protein mediators (e.g. avidin) are also amenable to hydrazidation. Enzyme hydrazides can be prepared in numerous ways as detailed above in section 1). The preferred embodiment of the invention is described here; however, this particular approach toward preparation in no way limits the use of other hydrazidation strategies which we have outlined in section 1).

Alkaline phosphatase (200 mg protein, 1.2 U/mg, Sigma Chem. Co.) is dissolved into a solution containing 1 g of adipic dihydrazide (12.5 ml, brought to pH 4.5). Water soluble carbodiimide (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide, WSC, 0.8 g) is added. During the course of the reaction, the pH is maintained between 4.5 and 5.5 by adding 1 N HCl. After 6 hr reaction at room temperature, the reaction mixture is maintained overnight at 4°C and then dialyzed against 0.9% (w/v) NaCl, and Tris buffer, pH 8.0 containing 1 mM MgCl₂ and 0.005% (w/v) NaN₃.

In the same manner, highly purified alkaline phosphatase (1200 U/mg) can be hydrazide modified using (per mg enzyme) 10 mg adipic dihydrazide and 2 to 10 mg WSA. 2. Staining Immobilized Glycoproteins with Enzyme Hydrazides

(1) Run protein sample on a standard SDS-PAGE (U.K. Laemmli, Nature (London) 227:680-6851970).

(2) At the end of run, blot the gel to nitrocellulose membrane filter as previously described (J.M. Gershoni, and G.E. Palade, Anal. Biochem. 124:396-4071982).

(3) Quench blot with 2% bovine serum albumin (BSA) in phosphate-buffered saline, pH 7.4 (PBS) 2-12 hr at room temperature or 37°C.

(4) The blot is then incubated in a 1-mM solution of sodium periodate 30 min at 4°C. (5) The oxidized blot is briefly rinsed in PbS ano then incubated 2 hr at room temperature with alkaline phosphatase-hydrazide (15 units/ml diluted a thousand-fold with a solution of 1% BSA in 50 mM Tris-HCl pH 7.4).

(6) The blot is thoroughly washed in PbS and then reacted with Napnthol As-Mx phosphate and Fast Red indol dye as described (H.Troyer (1980) Principles and Techniques of Histochemistry, Little, Brown and Co. Inc. Boston). Fast Violet, Fast Green or Fast Blue dyes may be substituted for Fast Reo in order to obtain the respective and desired color precipitate. The precipitate bands form at the areas containing immobilized sialoglycoproteins. In order to stain glycoproteins in general, high concentrations (> 25 mM) of periodate ano longer incubation times at different temperatures e.g. 1 hr at 25ºC) can be used. Fig. 1 demonstrates the labeling of erythrocyte membrane glycoproteins with alkaline phosphatase- hydrazide.

APPLICATIONS:

Enzyme hydrazides have oeen constructed for specific detection of glycoconjugates in cytochemical-microscopy, for gel electrophoretic analyses of glycoconjugates, for detection and analysis of glycoconjugates "blotted" onto nitrocellulose, nylon and other types of immobilizing matrices, etc. We have also indicated their suitability for the specific detection of macromolecules containing not only aldehydes out amines, hydroxyls, carboxyls, etc. (see section 3). Due to the fact that the enzyme hydrazides contain many hydrazide moieties per enzyme (or avidin or lectin or antibody; molecule, the system may be used to amplify the desired response or signal. Moreover, the enzyme hydrazides can be employed in other less straightforward procedures. For example, the described system can be used as a general protein stain. Since the enzyme hydrazide reacts specifically with aldehydes, glutaraldehyde fixed proteins react with this reagent as described in section 3.2. In addition, the sytem can be used for enzyme-conjugated immunoglobulins, i.e. the screening of monoclonal antibodies as well as immunocytochemical assays (e.g. ELISA) and various diagnostic kits which are based on coloriometric enzyme assays. As immunoglobulins are themselves glycoproteins and susceptible to periodate oxidation without losing their activity, enzyme hydrazides can easily be used for enzyme-conjugation of immunoglobulins. Glutaraldehyde-activated immunoglobulins can also interact with enzyme hydrazides for the preparation of conjugates.

In cases where intact cells are oxidized with periodate and then subjected to gel electrophoresis and blotting, enzyme-hydrazides can be used to demonstrate those proteins which were on the cell surface (and thus susceptible to the periodate oxidation.

The general approach here can also be used to immobilize proteins to solid supports or for crosslinking macromolecules or for preparing macromolecular conjugates.

Legend to the Figure:

Membrane proteins from human erthrocyte ghosts, separatee by SDS-PAGE, either stained directly with Coomassie Brilliant Blue (A) or PAS (B) or electrophoretically transferred to nitrocellulose memorane filters and stained for glycoconjugates using alkaline phosphatase-hydrazide (C).

Claims

US ClaimsCLAIMS:

1. A hydrazide derivative of an enzyme wherein the hydrazine or hydrazide moiety is linked to the enzyme molecule either directly or via a functionally inert spacer, via a functionally active molecule or via an antibody directed to the specific enzyme.

2. A derivative according to claim 1, wherein the hydrazine or polymeric form of hydrazide is directly covalently linked to the enzyme.

3. A hydrazide derivative according to claim 1, wherein the hydrazide is linked to the enzyme via a functional group not affecting enzymatic activity selected from amino, carboxyl, aldehydo or sulfhydryl groups of the enzyme.

4. A hydrazide derivative according to claim 1, wherein there is used a hydrazine, or a dihydrazide (i.e. either H₂NHNCONHNH₂ or of the general formula H₂NHNOC-(CH₂)_n-CONHNH₂ where 0<n>12) in the presence of a water soluble carbodiimide forming an intermediate link.

5. A hydrazide derivative according to claim 1 wherein an enzyme is linked via an antibody to the hydrazido group, or where the enzyme is linked via the avidin-biotin complex or via lectin-sugar interactions to the hydrazido moiety.

6. A hydrazide according to claim 1 wherein the enzyme is selected from alkaline phosphatases, acid phosphatase, 5'-nucleotidase, glucose 6-phosphatase, acetylcholine esterase, β-galactosidase, dopa-oxidase, horseradish peroxidase.

7. A hydrazido derivative according to claim 1, wherein an enzyme-hydrazido conjugate is linked to an amino-containing macromolecuie, to an aldehyde containing macromolecuie, to a carboxyl containing macromolecuie forming respectively compounds of the general formulas R-NHCO-enz, R-CH=N-NHCO-enz, R-CH₂NHNHCO-enz or R-CONHNH-enz wherein R is the residue of the macromolecuie.

8. A method for the detection and determination of glycoconjugates (either naturally occuring glycoconjugates or other macromolecules which have been glycosylated by chemical, biochemical or biological means) for gel electrophoretic analysis of such glycoconjugates and there blots thereof, for the microscopic localization of such glycoconjugates, for the detection of macromolecules containing amino or aldehyde groups, carboxy or vicinal hydroxy groups, for the screening of monoclonal antibodies, which comprises interacting same with a hydrazido-enzyme conjugate according to claim 1 and utilizing the enzyme thereof for visualization or other detection and determination.