Chemical specificity

Last updated August 26, 2023

Chemical specificity is the ability of binding site of a macromolecule (such as a protein) to bind specific ligands. The fewer ligands a protein can bind, the greater its specificity.

Specificity describes the strength of binding between a given protein and ligand. This relationship can be described by a dissociation constant, which characterizes the balance between bound and unbound states for the protein-ligand system.^[1] In the context of a single enzyme and a pair of binding molecules, the two ligands can be compared as stronger or weaker ligands (for the enzyme) on the basis of their dissociation constants. (A lower value corresponds to a stronger binding.)

Specificity for a set of ligands is unrelated to the ability of an enzyme to catalyze a given reaction, with the ligand as a substrate.^[1]

If a given enzyme has a high chemical specificity, this means that the set of ligands to which it binds is limited, such that neither binding events nor catalysis can occur at an appreciable rate with additional molecules.

An example of a protein-ligand pair whose binding activity can be highly specific is the antibody-antigen system.^[2] Affinity maturation typically leads to highly specific interactions, whereas naive antibodies are promiscuous and bind a larger number of ligands.^[3] Conversely, an example of a protein-ligand system that can bind substrates and catalyze multiple reactions effectively is the Cytochrome P450 system, which can be considered a promiscuous enzyme due to its broad specificity for multiple ligands. Proteases are a group of enzymes that show a broad range of cleavage specificities. Promiscuous proteases as digestive enzymes unspecifically degrade peptides, whereas highly specific proteases are involved in signaling cascades.^[4]

Basis

Binding

The interactions between the protein and ligand substantially affect the specificity between the two entities. Electrostatic interactions and Hydrophobic interactions are known to be the most influential in regards to where specificity between two molecules is derived from.^[5] The strength of these interactions between the protein and ligand often positively correlate with their specificity for one another.

The specificity of a binding process is strongly dependent of the flexibility of the binding partners. A rigid protein is very restricted in its binding possibilities. A flexible protein can adapt its conformation to a larger number of ligands and thus is more promiscuous. As the binding process usually leads to a rigidification of both binding partners in the complex, binding of a flexible protein usually comes with an entropic penalty. This is the main reason for the frequently found positive correlation of binding affinity and binding specificity. Antibodies show a strong correlation between rigidity and specificity.^[6]^[3] This correlation extends far beyond the paratope of the antibodies ^[7]

Catalysis

Enzyme specificity refers to the interactions between any particular enzyme and its corresponding substrate. In addition to the specificity in binding its substrates, correct proximity and orientation as well as binding the transition state provide an additional layer of enzyme specificity.

Types

Enzymes vary in the specificity of the substrates that they bind to, in order to carry out specific physiological functions. Some enzymes may need to be less specific and therefore may bind to numerous substrates to catalyze a reaction. On the other hand, certain physiological functions require extreme specificity of the enzyme for a single specific substrate in order for a proper reaction and physiological phenotype to occur. The different types of categorizations differ based on their specificity for substrates. Most generally, they are divided into four groups: absolute, group, linkage, and stereochemical specificity.

Absolute specificity

Absolute specificity can be thought of as being exclusive, in which an enzyme acts upon one specific substrate.^[8] Absolute specific enzymes will only catalyze one reaction with its specific substrate. For example, lactase is an enzyme specific for the degradation of lactose into two sugar monosaccharides, glucose and galactose. Another example is Glucokinase, which is an enzyme involved in the phosphorylation of glucose to glucose-6-phosphate. It is primarily active in the liver and is the main isozyme of Hexokinase.^[9] Its absolute specificity refers to glucose being the only hexose that is able to be its substrate, as opposed to hexokinase, which accommodates many hexoses as its substrate.

Group specificity

Group specificity occurs when an enzyme will only react with molecules that have specific functional groups, such as aromatic structures, phosphate groups, and methyls.^[10] One example is Pepsin, an enzyme that is crucial in digestion of foods ingested in our diet, that hydrolyzes peptide bonds in between hydrophobic amino acids, with recognition for aromatic side chains such as phenylalanine, tryptophan, and tyrosine. Another example is hexokinase, an enzyme involved in glycolysis that phosphorylate glucose to produce glucose-6-phosphate. This enzyme exhibits group specificity by allowing multiple hexoses (6 carbon sugars) as its substrate.^[11] Glucose is one of the most important substrates in metabolic pathways involving hexokinase due to its role in glycolysis, but is not the only substrate that hexokinase can catalyze a reaction with.

Bond specificity

A reaction that illustrates an enzyme cleaving a specific bond of the reactant in order to create two products Linkagespecificity.png — A reaction that illustrates an enzyme cleaving a specific bond of the reactant in order to create two products

Bond specificity, unlike group specificity, recognizes particular chemical bond types. This differs from group specificity, as it is not reliant on the presence of particular functional groups in order to catalyze a particular reaction, but rather a certain bond type (for example, a peptide bond).

Stereochemical specificity

Sugars containing alpha-glycosidic linkages Alphaglucan.jpg — Sugars containing alpha-glycosidic linkages

This type of specificity is sensitive to the substrate's optical activity of orientation. Stereochemical molecules differ in the way in which they rotate plane polarized light, or orientations of linkages (see alpha, beta glycosidic linkages). Enzymes that are stereochemically specific will bind substrates with these particular properties. For example, beta-glycosidase will only react with beta-glycosidic bonds which are present in cellulose, but not present in starch and glycogen, which contain alpha-glycosidic linkages. This is relevant in how mammals are able to digest food. For instance, the enzyme Amylase is present in mammal saliva, that is stereo-specific for alpha-linkages, this is why mammals are able to efficiently use starch and glycogen as forms of energy, but not cellulose (because it is a beta-linkage).

Determination

Specific equilibrium dissociation constant for formation of the enzyme-substrate complex is known as $k_{d}$ . It is used as a measure of affinity, with higher values indicating a lower affinity.

For the given equation (E = enzyme, S = substrate, P = product),

E+S{\overset {k_{1}}{\underset {k_{-}{1}}{\Longleftrightarrow }}}ES{\overset {k_{2}}{\Longleftrightarrow }}E+P

$k_{d}$ would be equivalent to $k_{-1}/k_{1}$ , where $k_{1}$ and $k_{-1}$ are the rates of the forward and backward reaction, respectively in the conversion of individual E and S to the enzyme substrate complex.

Information theory allows for a more quantitative definition of specificity by calculating the entropy in the binding spectrum.^[4]

Application to enzyme kinetics

The chemical specificity of an enzyme for a particular substrate can be found using two variables that are derived from the Michaelis-Menten equation. $k_{m}$ approximates the dissociation constant of enzyme-substrate complexes. $k_{cat}$ represents the turnover rate, or the number of reactions catalyzed by an enzyme over the enzyme amount. $k_{cat}$ over $k_{m}$ is known as the specificity constant, which gives a measure of the affinity of a substrate to some particular enzyme. Also known as the efficiency of an enzyme, this relationship reveals an enzyme's preference for a particular substrate. The higher the specificity constant of an enzyme corresponds to a high preference for that substrate.

Significance

Medical research relevance

Enzymatic specificity provides useful insight into enzyme structure, which ultimately determines and plays a role in physiological functions.^[12] Specificity studies also may provide information of the catalytic mechanism.

Specificity is important for novel drug discovery and the field of clinical research, with new drugs being tested for its specificity to the target molecule in various rounds of clinical trials. Drugs must contain as specific as possible structures in order to minimize the possibility of off-target affects that would produce unfavorable symptoms in the patient. Drugs depend on the specificity of the designed molecules and formulations to inhibit particular molecular targets.^[1] Novel drug discovery progresses with experiments involving highly specific compounds. For example, the basis that drugs must successfully be proven to accomplish is both the ability to bind the target receptor in the physiological environment with high specificity and also its ability to transduce a signal to produce a favorable biological effect against the sickness or disease that the drug is intended to negate.^[13]

Applications

Scientific techniques, such as immunostaining, depend on chemical specificity. Immunostaining utilizes the chemical specificity of antibodies in order to detect a protein of interest at the cellular level.^[14] Another technique that relies on chemical specificity is Western blotting, which is utilized to detect a certain protein of interest in a tissue. This technique involves gel electrophoresis followed by transferring of the sample onto a membrane which is stained by antibodies. Antibodies are specific to the target protein of interest, and will contain a fluorescent tag signaling the presence of the researcher's protein of interest.^[15]

Related Research Articles

In chemistry, biochemistry, and pharmacology, a dissociation constant is a specific type of equilibrium constant that measures the propensity of a larger object to separate (dissociate) reversibly into smaller components, as when a complex falls apart into its component molecules, or when a salt splits up into its component ions. The dissociation constant is the inverse of the association constant. In the special case of salts, the dissociation constant can also be called an ionization constant. For a general reaction:

Enzymes are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. Almost all metabolic processes in the cell need enzyme catalysis in order to occur at rates fast enough to sustain life. Metabolic pathways depend upon enzymes to catalyze individual steps. The study of enzymes is called enzymology and the field of pseudoenzyme analysis recognizes that during evolution, some enzymes have lost the ability to carry out biological catalysis, which is often reflected in their amino acid sequences and unusual 'pseudocatalytic' properties.

In biochemistry, a kinase is an enzyme that catalyzes the transfer of phosphate groups from high-energy, phosphate-donating molecules to specific substrates. This process is known as phosphorylation, where the high-energy ATP molecule donates a phosphate group to the substrate molecule. This transesterification produces a phosphorylated substrate and ADP. Conversely, it is referred to as dephosphorylation when the phosphorylated substrate donates a phosphate group and ADP gains a phosphate group. These two processes, phosphorylation and dephosphorylation, occur four times during glycolysis.

A protease is an enzyme that catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products. They do this by cleaving the peptide bonds within proteins by hydrolysis, a reaction where water breaks bonds. Proteases are involved in many biological functions, including digestion of ingested proteins, protein catabolism, and cell signaling.

In biochemistry, phosphorylation is the attachment of a phosphate group to a molecule or an ion. This process and its inverse, dephosphorylation, are common in biology. Protein phosphorylation often activates many enzymes.

In biochemistry, allosteric regulation is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme's active site.

A hexokinase is an enzyme that phosphorylates hexoses, forming hexose phosphate. In most organisms, glucose is the most important substrate for hexokinases, and glucose-6-phosphate is the most important product. Hexokinase possesses the ability to transfer an inorganic phosphate group from ATP to a substrate.

In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the binding site, and residues that catalyse a reaction of that substrate, the catalytic site. Although the active site occupies only ~10–20% of the volume of an enzyme, it is the most important part as it directly catalyzes the chemical reaction. It usually consists of three to four amino acids, while other amino acids within the protein are required to maintain the tertiary structure of the enzymes.

In biochemistry and molecular biology, a binding site is a region on a macromolecule such as a protein that binds to another molecule with specificity. The binding partner of the macromolecule is often referred to as a ligand. Ligands may include other proteins, enzyme substrates, second messengers, hormones, or allosteric modulators. The binding event is often, but not always, accompanied by a conformational change that alters the protein's function. Binding to protein binding sites is most often reversible, but can also be covalent reversible or irreversible.

<span class="mw-page-title-main">Glucokinase</span> Enzyme participating to the regulation of carbohydrate metabolism

Glucokinase is an enzyme that facilitates phosphorylation of glucose to glucose-6-phosphate. Glucokinase occurs in cells in the liver and pancreas of humans and most other vertebrates. In each of these organs it plays an important role in the regulation of carbohydrate metabolism by acting as a glucose sensor, triggering shifts in metabolism or cell function in response to rising or falling levels of glucose, such as occur after a meal or when fasting. Mutations of the gene for this enzyme can cause unusual forms of diabetes or hypoglycemia.

Drug design, often referred to as rational drug design or simply rational design, is the inventive process of finding new medications based on the knowledge of a biological target. The drug is most commonly an organic small molecule that activates or inhibits the function of a biomolecule such as a protein, which in turn results in a therapeutic benefit to the patient. In the most basic sense, drug design involves the design of molecules that are complementary in shape and charge to the biomolecular target with which they interact and therefore will bind to it. Drug design frequently but not necessarily relies on computer modeling techniques. This type of modeling is sometimes referred to as computer-aided drug design. Finally, drug design that relies on the knowledge of the three-dimensional structure of the biomolecular target is known as structure-based drug design. In addition to small molecules, biopharmaceuticals including peptides and especially therapeutic antibodies are an increasingly important class of drugs and computational methods for improving the affinity, selectivity, and stability of these protein-based therapeutics have also been developed.

Mixed inhibition is a type of enzyme inhibition in which the inhibitor may bind to the enzyme whether or not the enzyme has already bound the substrate but has a greater affinity for one state or the other. It is called "mixed" because it can be seen as a conceptual "mixture" of competitive inhibition, in which the inhibitor can only bind the enzyme if the substrate has not already bound, and uncompetitive inhibition, in which the inhibitor can only bind the enzyme if the substrate has already bound. If the ability of the inhibitor to bind the enzyme is exactly the same whether or not the enzyme has already bound the substrate, it is known as a non-competitive inhibitor. Non-competitive inhibition is sometimes thought of as a special case of mixed inhibition.

Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the biomolecule of interest; antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid binding interactions are frequently exploited for isolation of various biomolecules. Affinity chromatography is useful for its high selectivity and resolution of separation, compared to other chromatographic methods.

Non-competitive inhibition is a type of enzyme inhibition where the inhibitor reduces the activity of the enzyme and binds equally well to the enzyme whether or not it has already bound the substrate. This is unlike competitive inhibition, where binding affinity for the substrate in the enzyme is decreased in the presence of an inhibitor.

Affinity labels are a class of enzyme inhibitors that covalently bind to their target causing its inactivation. The hallmark of an affinity label is the use of a targeting moiety to specifically and reversibly deliver a weakly reactive group to the enzyme that irreversibly binds to an amino acid residue. The targeting portion of the label often resembles the enzyme's natural substrate so that a similar mode of noncovalent binding is used prior to the covalent linkage. Their usefulness in medicine can be limited by the specificity of the first noncovalent binding step whereas indiscriminate action can be utilized for purposes such as affinity labeling - a technique for the validation of substrate-specific binding of compounds.

Enzyme kinetics is the study of the rates of enzyme-catalysed chemical reactions. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction are investigated. Studying an enzyme's kinetics in this way can reveal the catalytic mechanism of this enzyme, its role in metabolism, how its activity is controlled, and how a drug or a modifier might affect the rate.

An enzyme inhibitor is a molecule that binds to an enzyme and blocks its activity. Enzymes are proteins that speed up chemical reactions necessary for life, in which substrate molecules are converted into products. An enzyme facilitates a specific chemical reaction by binding the substrate to its active site, a specialized area on the enzyme that accelerates the most difficult step of the reaction.

A ligand binding assay (LBA) is an assay, or an analytic procedure, which relies on the binding of ligand molecules to receptors, antibodies or other macromolecules. A detection method is used to determine the presence and extent of the ligand-receptor complexes formed, and this is usually determined electrochemically or through a fluorescence detection method. This type of analytic test can be used to test for the presence of target molecules in a sample that are known to bind to the receptor.

A protein–ligand complex is a complex of a protein bound with a ligand that is formed following molecular recognition between proteins that interact with each other or with other molecules. Formation of a protein-ligand complex is based on molecular recognition between biological macromolecules and ligands, where ligand means any molecule that binds the protein with high affinity and specificity. Molecular recognition is not a process by itself since it is part of a functionally important mechanism involving the essential elements of life like in self-replication, metabolism, and information processing. For example DNA-replication depends on recognition and binding of DNA double helix by helicase, DNA single strand by DNA-polymerase and DNA segments by ligase. Molecular recognition depends on affinity and specificity. Specificity means that proteins distinguish the highly specific binding partner from less specific partners and affinity allows the specific partner with high affinity to remain bound even if there are high concentrations of less specific partners with lower affinity.

<span class="mw-page-title-main">Competitive inhibition</span> Interruption of a chemical pathway

Competitive inhibition is interruption of a chemical pathway owing to one chemical substance inhibiting the effect of another by competing with it for binding or bonding. Any metabolic or chemical messenger system can potentially be affected by this principle, but several classes of competitive inhibition are especially important in biochemistry and medicine, including the competitive form of enzyme inhibition, the competitive form of receptor antagonism, the competitive form of antimetabolite activity, and the competitive form of poisoning.

References

1 2 3 Eaton, Bruce E.; Gold, Larry; Zichi, Dominic A. (1995-10-01). "Let's get specific: the relationship between specificity and affinity". Chemistry & Biology. 2 (10): 633–638. doi: 10.1016/1074-5521(95)90023-3 . PMID 9383468.
↑ Tanford, Charles (1968). "Chemical basis for antibody diversity and specificity". Accounts of Chemical Research. 1 (6): 161–167. doi:10.1021/ar50006a001.
1 2 Fernández-Quintero, Monica L.; Georges, Guy; Varga, Janos M.; Liedl, Klaus R. (2021). "Ensembles in solution as a new paradigm for antibody structure prediction and design". mAbs. 13 (1): e1923122. doi: 10.1080/19420862.2021.1923122 . PMC 8158028 . PMID 34030577.
1 2 Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Margreiter, Michael A.; Spitzer, Gudrun M.; Wallnoefer, Hannes G.; Liedl, Klaus R. (2013-04-18). "Cleavage Entropy as Quantitative Measure of Protease Specificity". PLOS Comput Biol. 9 (4): e1003007. Bibcode:2013PLSCB...9E3007F. doi: 10.1371/journal.pcbi.1003007 . ISSN 1553-7358. PMC 3630115 . PMID 23637583.
↑ Waldner, Birgit J.; Kraml, Johannes; Kahler, Ursula; Spinn, Alexander; Schauperl, Michael; Podewitz, Maren; Cruciani, Gabriele; Liedl, Klaus R. (2018). "Electrostatic recognition in substrate binding to serine proteases". Journal of Molecular Recognition. 31 (10): e2727. doi: 10.1002/jmr.2727 . PMC 6175425 . PMID 29785722.
↑ Fernández-Quintero, Monica L.; Loeffler, Johannes R.; Kraml, Johannes; Kahler, Ursula; Kamenik, Anna S.; Liedl, Klaus R. (2019). "Characterizing the Diversity of the CDR-H3 Loop Conformational Ensembles in Relationship to Antibody Binding Properties". Frontiers in Immunology. 9: 3065. doi: 10.3389/fimmu.2018.03065 . PMC 6330313 . PMID 30666252.
↑ Fernández-Quintero, Monica L.; Loeffler, Johannes R.; Bacher, Lisa M.; Waibl, Franz; Seidler, Clarissa A.; Liedl, Klaus R. (2020). "Local and Global Rigidification Upon Antibody Affinity Maturation". Frontiers in Molecular Biosciences. 7: 182. doi: 10.3389/fmolb.2020.00182 . PMC 7426445 . PMID 32850970.
↑ "Enzyme Specificity" (PDF). Archived from the original (PDF) on 2016-05-08.
↑ "GCK glucokinase [Homo sapiens (human)] - Gene - NCBI". www.ncbi.nlm.nih.gov. Retrieved 2016-06-12.
↑ "MSOE Center for BioMolecular Modeling -Protein Structure Jmol Tutorials">". cbm.msoe.edu. Retrieved 2016-05-19.
↑ Sener, A; Giroix, M H; Dufrane, S P; Malaisse, W J (1985-09-01). "Anomeric specificity of hexokinase and glucokinase activities in liver and insulin-producing cells". Biochemical Journal. 230 (2): 345–351. doi:10.1042/bj2300345. ISSN 0264-6021. PMC 1152624 . PMID 3902008.
↑ Pi, Na; Leary, Julie A (2004-02-01). "Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay". Journal of the American Society for Mass Spectrometry. 15 (2): 233–243. doi:10.1016/j.jasms.2003.10.009. PMID 14766290.
↑ "drug_receptor_theory [TUSOM | Pharmwiki]". tmedweb.tulane.edu. Retrieved 2016-06-11.
↑ Maity, Biswanath; Sheff, David; Fisher, Rory A. (2013-01-01). Immunostaining: detection of signaling protein location in tissues, cells and subcellular compartments. pp. 81–105. doi:10.1016/B978-0-12-407239-8.00005-7. ISBN 9780124072398. ISSN 0091-679X. PMID 23317899.{{cite book}}: |journal= ignored (help)
↑ Bass, J. J.; Wilkinson, D. J.; Rankin, D.; Phillips, B. E.; Szewczyk, N. J.; Smith, K.; Atherton, P. J. (2016-06-05). "An overview of technical considerations for Western blotting applications to physiological research". Scandinavian Journal of Medicine & Science in Sports. 27 (1): 4–25. doi:10.1111/sms.12702. ISSN 1600-0838. PMC 5138151 . PMID 27263489.

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[:0-1] 1 2 3 Eaton, Bruce E.; Gold, Larry; Zichi, Dominic A. (1995-10-01). "Let's get specific: the relationship between specificity and affinity". Chemistry & Biology. 2 (10): 633–638. doi: 10.1016/1074-5521(95)90023-3 . PMID 9383468.

[2] Tanford, Charles (1968). "Chemical basis for antibody diversity and specificity". Accounts of Chemical Research. 1 (6): 161–167. doi:10.1021/ar50006a001.

[fqm-3] 1 2 Fernández-Quintero, Monica L.; Georges, Guy; Varga, Janos M.; Liedl, Klaus R. (2021). "Ensembles in solution as a new paradigm for antibody structure prediction and design". mAbs. 13 (1): e1923122. doi: 10.1080/19420862.2021.1923122 . PMC 8158028 . PMID 34030577.

[Fuchs-4] 1 2 Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Margreiter, Michael A.; Spitzer, Gudrun M.; Wallnoefer, Hannes G.; Liedl, Klaus R. (2013-04-18). "Cleavage Entropy as Quantitative Measure of Protease Specificity". PLOS Comput Biol. 9 (4): e1003007. Bibcode:2013PLSCB...9E3007F. doi: 10.1371/journal.pcbi.1003007 . ISSN 1553-7358. PMC 3630115 . PMID 23637583.

[5] Waldner, Birgit J.; Kraml, Johannes; Kahler, Ursula; Spinn, Alexander; Schauperl, Michael; Podewitz, Maren; Cruciani, Gabriele; Liedl, Klaus R. (2018). "Electrostatic recognition in substrate binding to serine proteases". Journal of Molecular Recognition. 31 (10): e2727. doi: 10.1002/jmr.2727 . PMC 6175425 . PMID 29785722.

[6] Fernández-Quintero, Monica L.; Loeffler, Johannes R.; Kraml, Johannes; Kahler, Ursula; Kamenik, Anna S.; Liedl, Klaus R. (2019). "Characterizing the Diversity of the CDR-H3 Loop Conformational Ensembles in Relationship to Antibody Binding Properties". Frontiers in Immunology. 9: 3065. doi: 10.3389/fimmu.2018.03065 . PMC 6330313 . PMID 30666252.

[7] Fernández-Quintero, Monica L.; Loeffler, Johannes R.; Bacher, Lisa M.; Waibl, Franz; Seidler, Clarissa A.; Liedl, Klaus R. (2020). "Local and Global Rigidification Upon Antibody Affinity Maturation". Frontiers in Molecular Biosciences. 7: 182. doi: 10.3389/fmolb.2020.00182 . PMC 7426445 . PMID 32850970.

[8] "Enzyme Specificity" (PDF). Archived from the original (PDF) on 2016-05-08.

[9] "GCK glucokinase [Homo sapiens (human)] - Gene - NCBI". www.ncbi.nlm.nih.gov. Retrieved 2016-06-12.

[10] "MSOE Center for BioMolecular Modeling -Protein Structure Jmol Tutorials">". cbm.msoe.edu. Retrieved 2016-05-19.

[11] Sener, A; Giroix, M H; Dufrane, S P; Malaisse, W J (1985-09-01). "Anomeric specificity of hexokinase and glucokinase activities in liver and insulin-producing cells". Biochemical Journal. 230 (2): 345–351. doi:10.1042/bj2300345. ISSN 0264-6021. PMC 1152624 . PMID 3902008.

[12] Pi, Na; Leary, Julie A (2004-02-01). "Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay". Journal of the American Society for Mass Spectrometry. 15 (2): 233–243. doi:10.1016/j.jasms.2003.10.009. PMID 14766290.

[13] "drug_receptor_theory [TUSOM | Pharmwiki]". tmedweb.tulane.edu. Retrieved 2016-06-11.

[14] Maity, Biswanath; Sheff, David; Fisher, Rory A. (2013-01-01). Immunostaining: detection of signaling protein location in tissues, cells and subcellular compartments. pp. 81–105. doi:10.1016/B978-0-12-407239-8.00005-7. ISBN 9780124072398. ISSN 0091-679X. PMID 23317899.{{cite book}}: |journal= ignored (help)

[15] Bass, J. J.; Wilkinson, D. J.; Rankin, D.; Phillips, B. E.; Szewczyk, N. J.; Smith, K.; Atherton, P. J. (2016-06-05). "An overview of technical considerations for Western blotting applications to physiological research". Scandinavian Journal of Medicine & Science in Sports. 27 (1): 4–25. doi:10.1111/sms.12702. ISSN 1600-0838. PMC 5138151 . PMID 27263489.

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