REVIEW |
Correspondence to: Cornelis J.F. Van Noorden, Dept. of Cell Biology and Histology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. E-mail: c.j.vannoorden@amc.uva.nl
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Summary |
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Specific enzymes play key roles in many pathophysiological processes and therefore are targets for therapeutic strategies. The activity of most enzymes is largely determined by many factors at the post-translational level. Therefore, it is essential to study the activity of target enzymes in living cells and tissues in a quantitative manner in relation to pathophysiological processes to understand its relevance and the potential impact of its targeting by drugs. Proteases, in particular, are crucial in every aspect of life and death of an organism and are therefore important targets. Enzyme activity in living cells can be studied with various tools. These can be endogenous fluorescent metabolites or synthetic chromogenic or fluorogenic substrates. The use of endogenous metabolites is rather limited and nonspecific because they are involved in many biological processes, but novel chromogenic and fluorogenic substrates have been developed to monitor activity of enzymes, and particularly proteases, in living cells and tissues. This review discusses these substrates and the methods in which they are applied, as well as their advantages and disadvantages for metabolic mapping in living cells. (J Histochem Cytochem 49:14731486, 2001)
Key Words: synthetic substrates, living cells, enzyme histochemistry, metabolic mapping, fluorogenic substrates, chromogenic substrates
SPECIFIC ENZYMES play key roles in many pathophysiological processes. In particular, proteases are enzymes that are responsible for essential steps in many diseases (
Many enzymes, and particularly proteases, are present in cells and tissue compartments in an inactive form because they are either synthesized as precursors that have little if any catalytic activity and need post-translational activation or they are bound to an endogenous inhibitor. The inactive enzyme can be converted to its active form by proteolytic processing by specific proteases, autocatalysis, or by binding of co-factors or removal of inhibitors. Hence, large amounts of inactive and therefore not functional enzyme can be accumulated in a tissue compartment. However, the enzyme can become activated rapidly on demand. This can be achieved, for example, by an amplification loop, in which a small amount of the active protease can directly or indirectly activate its inactive precursor in a defined cell or tissue compartment, resulting in an exponential rate of activation to ensure that the protease can accomplish its function locally when required. Endogenous inhibitors are present in tissues to establish a threshold that regulates the concentration of active proteases in cells and tissues, thus keeping proteolysis under control (
Localization of the activity of an enzyme is traditionally performed at substrate concentrations that produce maximal amounts of colored or fluorescent final reaction product. These concentrations are usually high so that the maximal velocity of the enzyme (Vmax) is obtained. However, these high substrate concentrations are seldom present in vivo. Moreover, the affinity of an enzyme for its substrate(s) can also be under post-translational control (
Localization and quantification of the activity of enzymes in living cells and tissues can be performed by analysis of either the production or the consumption of fluorescent endogenous molecules, such as NADPH and NADH, or the formation of colored or fluorescent products generated from synthetic chromogenic or fluorogenic substrates using digital microscopy or flow cytometry. This setup allows quantitative monitoring of enzyme reactions in cells and tissues in time and space while the reaction proceeds. Although endogenous fluorescent molecules such as NAD(P)H, flavins, and porphyrins can be useful indicators of enzyme activity, such endogenous metabolites are limited in number and are not specific for a single enzyme. These molecules are both produced and consumed permanently in various enzymatic processes. Therefore, monitoring of these molecules in cells and tissues is not a good parameter of the activity of a specific enzyme. Consequently, the design, synthesis, and application of synthetic chromogenic and fluorogenic substrates are indispensable to identify specific enzymatic reactions in in vivo experiments. These substrates must meet a series of criteria for the application to living cells and tissues, such as the following.
When enzyme reactions are analyzed quantitatively, one should realize that kinetic parameters of enzymes can be seriously affected by various properties of the synthetic chromogenic or fluorogenic substrates. A number of these pitfalls are listed below.
All these phenomena can affect the kinetics of enzymes and can therefore complicate the analysis of the activity of enzymes by metabolic mapping. For example, for accurate determination of enzyme activity with substrates containing two sites of action for an enzyme, the two-step catalysis, the channeling effect of intermediate products and the intracellular substrate concentrations should be considered, as described by
This review discusses the advantages and disadvantages of fluorescent metabolites and chromogenic and fluorogenic substrates for their use in metabolic mapping in living cells and tissues on the basis of these criteria and considerations.
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Metabolic Mapping in Living Cells by Use of Endogenous Fluorescent Metabolites |
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A number of cellular metabolites are fluorescent. These fluorescent endogenous metabolites can be used to monitor enzyme reactions in living cells and tissues (Table 1). However, most of these metabolites must be excited with ultraviolet (UV) light, which rapidly damages living cells and tissues (
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The characteristics of autofluorescence are as follows:
Comparison of spectra of intact cells with spectra of known cell metabolites indicates that autofluorescence in cells arises mainly from intracellular NADH and riboflavin, flavin co-enzymes, and flavoproteins present in mitochondria. Co-enzymes fluoresce when in the reduced state (NAD(P)H) and do not fluoresce in the oxidized state (NAD(P)), whereas flavins fluoresce when in the oxidized state (FAD) and fluorescence disappears during reduction (FADH2). It is not known why autofluorescence in living cells varies so widely but, to a certain extent, intensities of autofluorescence reflect intracellular concentrations of NADH and FAD (
The spectra of the components NADH and riboflavin compare well with the spectra of autofluorescent cells and the metabolic activity in these cells.
Experiments with HeLa cells transfected with GFP-tagged histone 2B clearly demonstrated the phototoxic effects of excitation light. It was expected that a beta sheet surrounding the GFP fluorochrome would prevent energy transfer to surrounding molecules to enhance the quantum efficiency and thus would restrict phototoxic effects. However, transfected cells appeared to be much more vulnerable to phototoxic effects than untransfected cells, which resulted in cell-cycle arrest or cell death. These living cells can be imaged only during the entire cell cycle, when the total amount of excitation light is kept to an absolute minimum of approximately 10 J cm-2 (E. Manders, personal communication). Manders' experiments demonstrated the dose-dependent relationship between the amount of excitation light and cell damage. However, the type of fluorophore also plays an important role.
Exposure of cells to stressful conditions, such as excitation, triggers stress responses. Solar UV light is a major source of environmental stress for mammalian cells () receptor family (
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Synthetic Chromogenic Substrates for Metabolic Mapping in Living Cells |
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Tetrazolium salt methods are established methods for the localization of the activity of dehydrogenases, reductases, and oxidases. Enzyme-catalyzed oxidation of the substrate liberates protons that are then transferred to a tetrazolium salt such as (tetra)nitro BT as final electron acceptor. In this way, a water-insoluble formazan is produced (
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D,T-diaphorase that can use either NADH or NADPH as substrate was demonstrated in living fish hepatocytes as well (
Reduction of tetrazolium salts has been used as test for viability of eukaryotic and prokaryotic cells. Although this approach is not specific for a particular dehydrogenase or reductase, it has been used successfully for subcellular localization of reducing enzyme systems in intact human hepatoma cells (
In conclusion, chromogenic substrates thus far have been only rarely exploited for metabolic mapping in living cells and tissues, but this approach is promising, especially for high-throughput screening of effects of potential drugs on living cells, as absorbance measurements are simpler than fluorescence measurements.
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Synthetic Fluorogenic Substrates for Metabolic Mapping in Living Cells |
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Synthetic fluorogenic substrates can be used for determination of viability of cells, but also for metabolic mapping. Various fluorogenic substrates have been developed, especially for hydrolytic enzymes such as proteases. Here we discuss synthetic fluorogenic substrates that have been applied to living cells and tissues (Table 3).
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Fluorochromes Used in Synthetic Fluorogenic Substrates
Fluorogenic substrates are usually esters or ethers of phenolic fluorophores; 7-hydroxy-4-methylcoumarin (ß-methylumbelliferone) and its analogues fluorescein and resorufin are the most common examples of fluorophores used in fluorogenic substrates (
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Formation of esters or ethers of phenolic fluorophores results in a shift to shorter wavelengths of absorbance and either partial or total quenching of the long-wavelength fluorescence of the fluorophore. Fluorescein-based substrates are not fluorescent because quenching is complete as the dye is converted into a non-fluorescent colorless lactone by the formation of two ether or ester bonds. Therefore, these substrates do not cause background fluorescence and are among the most sensitive fluorogenic substrates known. Fluorescein is water-soluble, and free fluorescein can be retained in living cells for at least a few minutes at room temperature (
Cytochrome P450 activity in living cells can be analyzed using the 7-ethoxyresorufin-O-deethylase (EROD) assay based on the formation of fluorescence of resorufin (
Activity of cathepsin K was localized by
A unique fluorogenic substrate for phosphatases, 2-(5'-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)- quinazolinone or ELF-97-phosphate, has recently been developed (
Rhodamine-based fluorogenic dipeptidide substrates have been synthesized by
In contrast, coumarin-based substrates are less useful than rhodamine-based substrates because suboptimal conditions for detection of the fluorophore must be used to maximize spectral differences between substrate and product. Furthermore, a structural change in the aminocoumarin moiety does not take place when the substrate is cleaved and, therefore, coumarin-based substrates are less useful than rhodamine-based substrates (see also below). Recently, a new type of fluorogenic substrate for proteases has been synthesized based on the leaving group cresyl violet (
Casein conjugates of two BODIPY dyes, one named BODIPY fluorescein (FL) and the other BODIPY Texas red (TR), have been developed by
DQ-BSA is also based on the BODIPY fluorochrome. BODIPY FL is conjugated to BSA at a high molar ratio. The resulting conjugate is self-quenched by fluorescence energy transfer between neighboring BODIPY molecules. Although this substrate is not membrane-permeable, it can be internalized by forming a complex with anti-BSA that is taken up by the Fc receptor of macrophages (
The fluorogenic substrates for caspases in Table 3 are based on peptides of 18 amino acids containing caspase cleavage sequences, with two identical fluorophores covalently attached near their termini. Such substituted peptides are assumed to have an oval-shaped structure in solution due to the formation of intramolecular excitonic H-dimers between the fluorophores (
Substrates have been designed for specific enzymes, such as cathepsin D, which is overexpressed in a number of cancers (
The small molecular substrates listed in Table 3 are not very fluorescent themselves, but the internally quenched macromolecular substrates in Table 3 may have the advantage that fluorescence is negligible and therefore sensitivity of the enzymatic assay is higher due to an increased signal-to-noise ratio. On the other hand, when the substrate is also fluorescent but with spectral characteristics that are different from those of the product, measurement of intracellular substrate concentrations is possible.
Enzyme products that can be excited in the red or near-infrared region of the spectrum are, in principle, the best for in vivo imaging of enzyme activity because the excitation light is less harmful than light of shorter wavelenghts.
In the near future, frequency resonance energy transfer (FRET)-based substrates will become available for analysis of enzyme activity in living cells. FRET-based substrates should be synthesized in such a way that two different fluorophores, of which the emission peak of one overlaps with the excitation peak of the other, are located in close proximity at opposite sides of a bond susceptible to enzymatic cleavage. Preferably, these two fluorophores have a large Stoke's shift. Excitation of the fluorochrome with excitation and emission peaks at shorter wavelengths can then, in theory, result in enhanced fluorescence of the second fluorochrome, with excitation and emission peaks at longer wavelengths when the substrate is not enzymatically processed. When the substrate is cleaved, the FRET phenomenon disappears and fluorescence of the fluorochrome with excitation and emission peaks at shorter wavelengths appears. For example, when the fluorophores Alexa Fluor 488 and rhodamine are combined in such a FRET-based substrate, excitation of the Alexa Fluor 488 fluorophore in the intact substrate results in enhanced emission of rhodamine fluorescence, which is a measure for the local (intracellular) substrate concentration. Alexa Fluor 488 fluorescence itself is a measure of the amount of substrate processed. Therefore, FRET-based substrates will enable measurements of both the amount of product generated by enzymatic activity and the intracellular concentration of the substrate, even in subcompartments of cells. The use of this type of substrate would solve the problem of estimating local substrate concentrations in cells or cell compartments to calculate accurately Vmax and Km values for enzymes in living cells. Labeling peptide sequences with two different dyes has been described by
The FRET phenomenon can also be useful to demonstrate specificity of a substrate for an enzyme. When the enzyme of interest is tagged with, e.g., green fluorescent protein (GFP) by transfection, co-localization of the enzyme and the enzyme product containing a fluorophore that has an excitation peak that overlaps the emission peak of GFP may result in the FRET phenomenon.
In conclusion, fluorophores with high fluorescence quantum yield should be selected for incorporation into synthetic fluorogenic enzyme substrates to obtain sufficient sensitivity to analyze enzyme reactions at physiological substrate concentrations. Substrates that contain fluorophores with excitation peaks in the red or infrared region of the spectrum are the substrates of choice. Moreover, kinetic parameters of the enzyme for the synthetic fluorogenic substrate should resemble that for its natural substrate(s), as explained below. In addition to small molecular fluorogenic substrates, macromolecular substrates containing quenched fluorophores are useful for analysis of specific activity of enzymes in living cells and tissues, and the concept of FRET-based fluorogenic substrates is intriguing.
Reactivity of Synthetic Fluorogenic Substrates
The ability of an enzyme to discriminate among many potential substrates is an important factor in maintaining organization of most biological functions in the biocomplexity of cells and tissues. Although substrate selection can be regulated at many levels in a biological context, such as spatial and temporal localization of enzyme and substrate, concentrations of enzyme and substrate, and requirement of co-factors, substrate specificity at the enzyme active site is the overriding principle that determines the turnover of a substrate (
Rhodamine-based substrates exhibit a wide range of specificity constants. Amino acids in the P2 position in dipeptide substrates determine specificity in a large part. Comparison of the kinetic constants of plasmin or thrombin for the best dipeptide substrates with those for (Cbz-Arg-NH)2rhodamine 110 indicates that the large increases in Kcat/Km obtained by extending the single amino acid substrate with an appropriate P2 residue in a dipeptide substrate are primarily the result of a large increase in Kcat and a decrease in Km. Therefore, the specificity of proteinases for synthetic substrates depends to a great extent on interactions between amino acids in the active site of a protease and amino acid residues in the peptide substrate. Because occupation of the P2 position does not increase specificity of coumarin thiolester-based substrates as much as it increases specificity of rhodamine-based substrates, selectivity can be much greater with rhodamine-based substrates. Therefore, rhodamine-based substrates are in principle more useful to detect selectively protease activity in living cells and tissues.
In conclusion, characterization of substrate specificity of an enzyme provides useful information for the dissection of complex biological pathways and also provides the basis for the design of selective substrates and potent inhibitors to study enzyme activity.
Localization of Final Fluorescent Reaction Product
When a fluorescent final reaction product of an enzyme accumulates at a certain site, this does not automatically imply that it has been produced in that site. Chemical properties of the fluorophore have effects on its intracellular localization. The charge of a fluorophore, such as rhodamine, can lead to accumulation in mitochondria. However, the charge of a molecule depends on its pKa value. For example, rhodamine 110 has a pKa of 4.3 at physiological pH 7.2, and one in a thousand rhodamine molecules is in the protonated acidic state with a positive charge. In the protonated state, rhodamine 110 accumulates in mitochondria as a result of the intramitochondrial electric potential (
In conclusion, chemical properties of fluorogenic substrate and fluorescent product must be taken into account for proper localization of enzyme activity in living cells and tissues.
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Conclusions |
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The present overview of methods for detection of enzyme activity in living cells and tissues indicates our limited knowledge of molecular interactions that take place during incubation and recording of the formation of a colored or fluorescent reaction product. Metabolic mapping in living cells and tissues on the basis of endogenous fluorescent metabolites has only limited applications. Concentrations of these metabolites do not reflect the activity of a specific enzyme and these metabolites must be excited with UV light, which is detrimental to living cells and tissues. Excitation light of short wavelengths is far more damaging to cells because the energy content is higher than that of longer wavelengths. Moreover, UV light can induce activation of signal transduction pathways within minutes, leading to profound alterations in cellular metabolism.
Chromogenic substrates thus far have rarely been exploited for metabolic mapping in living cells and tissues, but this approach is promising, especially for high-throughput screening of effects of potential drugs on living cells, because absorbance measurements are far more simple than fluorescence measurements.
Important criteria for the selection of fluorophores to incorporate into synthetic fluorogenic substrates are a highfluorescence quantum yield to obtain sufficient sensitivity to analyze enzyme reactions at physiological substrate concentrations. Substrates that contain fluorophores with excitation peaks in the red or infrared region of the spectrum are the substrates of choice. Moreover, kinetic parameters of the enzyme for the synthetic fluorogenic substrate should resemble those for its natural substrate(s). In addition to small molecular fluorogenic substrates, macromolecular substrates containing quenched fluorophores are useful for analysis of specific activity of enzymes in living cells and tissues, and the concept of FRET-based fluorogenic substrates is promising.
Intrinsic chemical properties of fluorophores in synthetic substrates have a strong effect on their detection and also on the reactivity of the substrate. These phenomena may be due to many factors, such as sterical hindrance or different chemical properties of the fluorophores used. The specificity exhibited by many enzymes depends, to a large extent, on the interaction of subsite amino acids in the active site of the enzyme. Specificity can be characterized with the use of synthetic substrates by studying variations in the specificity constant on substitution or alteration of single amino acid residues in the substrates. Furthermore, localization of fluorophores in living cells at the site of enzyme activity is a major issue. Addition of chemical anchors to a fluorophore may improve localization properties.
Received for publication May 15, 2001; accepted July 11, 2001.
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