Chromogenix Chromogenic substrate molecules images

Chromogenix chromogenic substrate S-2222™ for Factor Xa	Chromogenix chromogenic substrate S-2238™ for Thrombin	Chromogenix chromogenic substrate S-2251™ for Plasmin	Chromogenix chromogenic substrate S-2288™ for tPA
Chromogenix chromogenic substrate S-2302™ for Plasma Kallikrein	Chromogenix chromogenic substrate S-2366™ for APC Resistance	Chromogenix chromogenic substrate S-2403™ for Plasmin	Chromogenix chromogenic substrate S-2765™ for Factor Xa

View our chromogenic substrates

The majority of the Chromogenix substrate library has an Arginine (Arg or R) group at the P1 position (the amino acid position that occurs at the preferred cleavage site). Why is this?

The Chromogenix line is geared toward the proteins involved in hemostasis. These are a group of proteolytic enzymes that comprise the serine proteases, which cleave mainly at the C-terminal side of the basic amino acids arginine or lysine. The peptides at the P2, P3, and P4 positions contribute to the substrate’s specificity. Note that the substrates for plasmin cleave at a lysine group. Other protease groups are aspartic proteases (like pepsin), metallo proteases, and cysteine proteases (which include caspases, with an asp cleavage site).

What if the reconstituted substrate has some precipitate or is cloudy?

The substrate solution is usually prepared with sterile water, but sometimes they may not dissolve properly. Sonication may help, or substrates with low solubility in water can be dissolved in DMSO, then diluted in water. The final DMSO concentration should preferably not exceed 10% in the reaction mixture. It should be noted that stability in DMSO is decreased, as it also is with alkaline buffers.

Why is pNA the leaving group on all of the Chromogenix substrates?

A good chromophore must be readily cleaved by and dissociated from the enzyme. The color must be strong to allow detection of low enzyme activities, but  should not interfere with the color of other reactants or impurities in the reaction mixture. It should be water soluble and have low toxicity. The chromophore para-nitroaniline (pNA) fulfills most of these requirements. It is therefore the most common choice of chromophore.

Why are the reactions measured at 405 nm?

The absorption intensity is expressed by the Beer-Lambert law, A = e x c x l, where A is absorbance, c is molar concentration, l is the path length of sample cell (usually 1 cm), and e is the extinction or molar absorptivity coefficient. The absorption spectrum of the substrate versus the chromophore, pNA (the chromogenic leaving group) is the reason for reading the reactions at 405 nm. The absorbance maximum of the unhydrolyzed, intact substrate is 316 nm and 380 nm for pNA. Although the difference between substrate and product is maximal at 385 nm, at 405 nm, there is less background reading, and the absorbance of the substrate is still less than 1% of that of an equimolar amount of pNA.

What aspects must a scientist consider when choosing the best chromogenic substrate?

Synthetic substrates are very sensitive; they can detect very low enzyme activities and are often more sensitive than a corresponding natural substrate. On the other hand, they can be less selective, or, have less discrimination in their reactivities toward related enzymes compared to the natural substrate. There are steps a scientist can take to maximize sensitivity and specificity. If the specificity of the enzymatic activity to be measured is known then a substrate selectivity table which shows the cross-reactivity of the substrates with different enzymes, and the kinetic data, such as that provided in the Chromogenix catalog, can be helpful. If the specificity of the enzyme is unknown, a screening procedure can be applied. This involves comparing the rate of hydrolysis obtained with different substrates. The presence of contaminating enzymes must also be taken into account. To eliminate interference, an inhibitor can be introduced, the sample can be further diluted, or conditions can be found where the relative activities are optimized. For instance, S-2222™ is selective for FXa, but also for trypsin. If a researcher wants to measure FXa, s/he can add an inhibitor to trypsin, such as soybean trypsin inhibitor. Temperature, pH, buffers, and ionic strength can all affect the rate of hydrolysis and must be considered. Substrate concentration is also important, and a concentration of 2 x Km is usually appropriate. A good substrate has a low Km, meaning maximum reaction velocity is achieved at a low substrate concentration. In other words, the enzyme has a high affinity for the substrate. A high Kcat is also desired, which means the enzyme has a high turnover rate with the substrate (fast reaction).

Define a katal.

One katal (kat) is the amount of enzyme that converts one mole of substrate per second. Activated enzymes from Chromogenix such as FXa and thrombin are measured in nkat. 1 nkat = 1 x 10^-9 mol of product released per second.

What is a chromogenic substrate composed of?

Chromogenic substrates are peptides that react with proteolytic enzymes under the formation of color. Chromogenic substrates are made up of a protecting group, amino acid residue(s), side-chain modification if applicable, and the chromophore. The stereochemistry of some substrates may be designated. For example, in the Chromogenix substrate S-2222™, the protecting group is a benzoyl group, the amino acid residue is Ile (isoleucine – a non-polar hydrophobic amino acid), the side chain modification is Glu(g-OR)- where R is 50% H (hydrogen) and CH₃ (methyl group). The P2 and P1 amino acid residues are Gly and Arg, respectively, and the chromophore is pNA (para-nitroaniline).

Define an enzyme and a substrate.

Enzymes are proteins that catalyze most of the chemical reactions that take place in the body. The chemical compound upon which the enzyme exerts its catalytic activity is called a substrate. Proteolytic enzymes degrade their substrates, proteins and peptides, by hydrolyzing one or more peptide bond(s). For information on enzyme kinetics, see the Chromogenix Catalog or contact Diapharma Group, Inc. at info@diapharma.com.

Are substrates available in alternate sizes such as 5mg, 500mg, 1g, etc?

Please inquire at info@diapharma.com.

What substrate research methods are available?

• Chromogenix Chromogenic Substrate S-2222 Trypsin
• Chromogenix Chromogenic Substrate S-2222 Tissue Factor Pathway Inhibitor, type – I (TFPI)
• Chromogenix Chromogenic Substrate S-2238 Antithrombin
• Chromogenix Chromogenic Substrate S-2238 Antithrombin (anti-FIIa)
• Chromogenix Chromogenic Substrate S-2288 Proteolytic activity
• Chromogenix Chromogenic Substrate S-2238 Thrombin
• Chromogenix Chromogenic Substrate S-2228 Tissue Plasminogen Activator (t-PA)
• Chromogenix Chromogenic Substrate S-2251 Plasmin
• Chromogenix Chromogenic Substrate S-2302 Kallikrein
• Chromogenix Chromogenic Substrate S-2302 Kallikrein Inhibitor
• Chromogenix Chromogenic Substrate S-2302 Prekallikrein
• Chromogenix Chromogenic Substrate S-2366 Hirudin
• Chromogenix Chromogenic Substrate S-2765 Factor X

• Diapharma Chromogenic Substrate CS GK (substitute for discontinued Chromogenix S-2266) Kallikrein in urine
• Diapharma Chromogenic Substrate CS UK (substitute for discontinued Chromogenix S-2444) Urokinase
• Diapharma Chromogenic Substrate CS PSA (KLK3) (substitute for discontinued Chromogenix S-2586) Chymotrypsin

Historically, the development of a new chromogenic substrate for a specific protease has always been accompanied by the release of method sheets where the application and the methodology for a particular use were described in detail. Some methods are simple chromogenic assays where a buffer and the substrate are the only reagents to be used (i.e. proteolytic activity). Other methods instead require the use of additional compounds, which are or have been commercially available from Chromogenix or elsewhere, and consist of more reaction steps. These protocols were validated in laboratories according to the equipment and the reagents available at the time. In several cases they have been adopted in research, quality control, or routine laboratories and some of them later became Chromogenix kits now present in our product range.

During the last 20 years, the so-called Method Sheets have been taken as the starting point by several scientists, for the development of assays for particular applications, or studies. In some cases the experimental conditions have been changed according to the particular need of the investigation being done. These methods are now presented in a different form: “Research Methods”. The intention here is to provide assay protocols that are not available as kits, but complementary to our product range. In the following list, you can find the methods developed by Chromogenix for several assays. They have to be considered as general guidelines or basic tools for the development of your own assays, some of them requiring a validation within your laboratory with respect to the reagents and equipment used.

For each method, you can find an updated Bibliography with references, where the method has been used, like as originally described or with modifications. This information should facilitate and accelerate the development of the best test protocol. If you do not have that specific journal issue in your laboratory, you can visit MEDLINE. You can search for particular articles, print the abstract and order the original copy through LOANSOME DOC (and receive the document through your local library). At the same time, on our web site you have the possibility to be updated on the new products from Chromogenix.

Theoretical basis for calculation

The hydrolysis of the chromogenic peptide substrate by the proteolytic enzyme follows in general the Michaelis-Menten kinetics. This means that, if the substrate is present at a sufficiently high concentration or if a comparatively small fraction of the substrate is hydrolized, the rate of product (color) formation is proportional to the activity of the enzyme. The rate of pNA formation, i.e. the increase in absorbance per second, is measured photometrically at 405 nm. At this wavelength the extinction coefficient of pNA is

9600 mol ^-1 • l • cm ^-1

The enzymatic activity can be quantified in two ways:

1. By comparing the activity of an enzyme with that of a standard preparation, which is defined in terms of a specified number of units set by an international or national authority or society (WHO, NIH etc.), or by the activity present in 1 ml of activated pooled normal plasma (Plasma Equivalent Unit = PEU). The standardization is performed by using at standard curve obtained with at least five different concentrations, each performed in duplicate. The standard material must be of the same kind and of the same quality as the sample which is to be measured. This may be still more important for a secondary or domestic standard.
2. By measuring the amount (mol) of substrate split, or rather product formed per unit time (absolute activity).

One unit of enzymatic activity, katal (kat) is defined as the amount of activity that converts one mole of substrate per second under standardized conditions. Such conditions as type of substrate, substrate concentration, buffer, pH, ionic strength and temperature should be given along with unit.

Thus, 1 nkat gives a conversion rate of:

1 x 10^-9 mol/sec = 60 x 10^-9 mol/min

If the total (measuring) volume used is V (ml), the increase in concentration per minute caused by 1 nkat is

If the absorbance is measured at 405 nm, in a 1 cm cuvette the difference in extinction coefficient is

e = 9600 mol-1 • l

The increase in absorbance/min can then be calculated by using Lambert-Beer’s law:

A = e x C

Thus, 1 nkat gives:

or

By using a sample volume v (ml):

For the end-point method, the incubation time t (min) with substrate is taken into account by the following formula:

According to nomenclature, one unit (U) is the amount of enzyme activity that converts one mol of substrate per minute under standardized conditions. By using the above formulas the units are:

Substrate kinetic and selectivity tables

Kinetic data for the chromogenic substrates available from Chromogenix. Suitable chromogenic substrates are listed for a number of serine proteases, most of them part of the cascade systems in blood. Some of the substrates are cleaved by more than one enzyme although at different rates. The kinetic analyses of the enzymatic cleavage of pNA from the substrates were performed under strictly standardized conditions using the clinical chemistry analyzer Cobas Mira S.

A stable, well-defined temperature is vital for all enzyme kinetic studies and in this study all reactions were performed at 37°C. A suitable buffer was chosen for each enzyme and the pH value given in the compilation is the value to which it was adjusted at 25°C. Note that the pH value of Tris buffers decreases as the temperature increases, at the rate of approximately 0.1 unit per °C (50 mM Tris-HCl). The kinetics of the reaction was followed spectrophotometrically by measuring the change in absorbance over time, ΔA/min. To ensure the highest precision, ΔA/min was measured at four different substrate concentrations. Insertion of the ΔA/min values into Eq. 18, followed by linear regression gave Km, kcat and Vmax for the reaction.

View Substrate Kinetic Tables

View Substrate Selectivity Tables

Describe chromogenic substrates in practice

Measurements made using chromogenic substrates reflect enzyme activity. Often it is more important to have knowledge about the activity of an enzyme than of the amount or mass – the quantity recorded in an immunological assay. Synthetic substrates are very sensitive, i.e. they can detect very low enzyme activities. They are in fact often more sensitive than a corresponding natural substrate.

This ability of chromogenic substrates to detect low enzyme concentrations makes them useful in, for example, the search for the presence of certain enzyme activities either in research or in quality control procedures. Sometimes there is a lack in correspondence between a natural and a chromogenic substrate in their responses to a certain enzyme preparation. For example, thrombin that has been partly degraded through autohydrolysis (ß-thrombin) reacts just as well with its chromogenic substrate as does the native form of thrombin (α-thrombin) while only native thrombin reacts with the natural substrate fibrinogen.

A chromogenic substrate is less selective, i.e. it has less discrimination in its reactivity towards related enzymes compared to the natural substrate. However, this lack of absolute selectivity can be compensated for when setting up chromogenic substrate assays. This is done by the proper selection of type of buffer, pH, relative concentrations of sample and reagents, addition of inhibitors, and/or choice of activator or incubation times. When presented with the opportunity of using one or more chromogenic substrates in a particular experimental setting for which there is no existing method, there are a few considerations that are worthwhile to make.

Substrate

If the specificity of the enzymatic activity to be measure-red is known then comprehensive overviews such as the Selectivity Tables will serve as a guide in selecting a proper substrate. The local distributor of Chromogenix products may also be contacted for advice on the choice of substrate(s). If the specificity of the enzyme is unknown, a screening procedure can be applied. When different substrates are available, such screening of the enzyme specificity can be carried out by comparing the rate of hydrolysis or pNA-generation obtained with the different substrates. Unless certain experience is available to the investigators it is usually advisable to discuss the plan and/or the result with Chromogenix. Advice on the next step can thus be given concerning either continued screening or the selection of a particular substrate that is suitable in the planned investigation.

Contaminating enzymes

If the sample to be tested with a chromogenic substrate contains more than one enzyme that may react with the same substrate, there are a number of measures that can be taken in order to eliminate the interfering/ contaminating activity. A natural or synthetic inhibitor can be introduced, the sample can be further diluted or conditions can be found (different pH and/or buffer) where the relative activities of the present enzymes are optimized. Such considerations can be based on the information below concerning temperature, pH, buffer and ionic strength.

Temperature

The rate by which the chromogenic substrate is cleaved is highly dependent on the temperature. It is therefore important to know at what temperature(s) a particular method is applicable – it may be at room (ambient) temperature, 25, 30, or 37 °C. An increase in temperature of 1 °C causes an increase in the reaction velocity of 2.5-7.5%. The temperature thus must be kept constant during the measurement and if results from different experiments are to be compared they must be performed at the same temperature. It is advisable to run the reactions in thermostated cuvettes and to use preheated stock solutions.

pH

Both Km and kcat are dependent on the pH. This means that kinetic calculations can only be made using results obtained at the same pH. Usually, the enzyme activity is measured at the pH optimum for the proteolytic activity of the enzyme. However, when several proteases are present in the same solution, as, e.g. when the sample is from plasma, it is not always advantageous to search for the pH that gives the maximum reactivity of the enzyme under investigation. Instead it is better to choose a pH where other serine proteases that may compete for the substrate have relatively lower levels of activity.

Buffers

The buffer medium and the concentration of buffer substances must be well defined. Usually Tris-HCl is used since the pKa of Tris buffer is 8.1 (25 °C), which makes it suitable for measurements at pH values between 7.3-9.3, where most of the serine proteases show maximal activities. Furthermore, this buffer is stable – it can even be autoclaved. Tris-imidazole has also been used, but is not to be recommended as imidazole is known to slightly inhibit certain proteases such as trypsin and plasmin.

Ionic strength and other additives

The appropriate ionic strength is usually obtained by adjusting the concentration of NaCl. Further substances that it may be necessary to add are CaCl2 (when Ca-dependent enzymes are studied), NaN3 (or other bactericidal agents) to prevent bacterial growth and polyethylene glycol or Tween 80 to prevent adsorption of the enzymes to the reaction vessel walls.

Substrate handling

The substrate solution is usually prepared by adding sterile water to the dry powder. Chromogenic substrates with low solubility in water can be dissolved in DMSO (dimethyl sulfoxide) and then diluted in water. The final DMSO concentration should preferably not exceed 10% in the reaction mixture. Chromogenic substrates dissolved in sterile water are stable for more than 6 months in the refrigerator (2 – 8 °C) and for several weeks at room temperature (25 °C). The stability is considerably reduced in alkaline buffers. Furthermore, contamination by microorganisms and exposure to light for longer periods should be avoided. The substrate concentration should be chosen so that linear kinetics is obtained. A substrate concentration of twice the Km (2 x Km ) is usually appropriate.

What is enzyme specificity and substrate selectivity?

Specificity is a property of the enzyme and describes how restrictive the enzyme is in its choice of substrate; a completely specific enzyme would have only one substrate.

The specificity of the serine proteases is usually not very high since they have similar active sites and act through the same proteolytic mechanism.

Consequently, a single serine protease may act on various substrates although at different rates. How the substrate fits the active site of the enzyme is of crucial importance to the outcome of the enzyme-substrate reaction. The bond to be cleaved must have a specific orientation relative to the amino acid side chains of the catalytic triad. The most important factor governing the fit of a substrate for an enzyme is the amino acid sequence around the bond to be cleaved.

Trypsin cleaves amides and esters of the basic amino acids arginine and lysine. Thrombin has a similar preference, but is more specific for arginine than for lysine.

Selectivity is a property of the substrate and indicates the degree to which the substrate is bound to and cleaved by different enzymes. The best measure for selectivity is given by the ratio kcat/Km. Synthetic substrates are considerably smaller than the natural substrates and can usually be cleaved by more than one enzyme, i. e. synthetic substrates are not completely selective. The explanation for this is that large substrates such as fibrinogen not only interact with the active site but also with exterior domains of the enzyme. Such interactions allow substrates to discriminate between different serine proteases and fibrinogen thus becomes highly selective for thrombin.

Selectivity Tables

The selectivity data of the table have been compiled to permit the investigator to understand how a contaminating enzyme would influence the enzyme-substrate reaction under study. Another way of expressing this is to say that the table shows the relative reactivities of two or more enzymes on one particular substrate. The table should be read horizontally. Each row represents the reactivity of a substrate designated for use with a particular enzyme, indicated to the left, relative to other relevant enzymes.

Example: The set of data in the top row shows the relative reactivity of the thrombin substrate S-2238™ with various enzymes. All the experiments were performed using the same buffer, i.e. the one most appropriate for the reaction between thrombin and chromogenic substrate S-2238™. In addition, the substrate concentration was always the same, or 2 x Km for the reaction of chromogenic substrate S-2238™ with thrombin. The concentrations of the different enzymes are given in Table 2 and are related to the plasma concentration of the corresponding zymogen. The reactivity of chromogenic substrate S-2238™ with thrombin, measured as the time-dependent increase in absorbance (ΔA/min), is given the value 100% (the actual value of ΔA/min is given in brackets). The reactivities of chromogenic substrate S-2238™ with the enzymes FXa, FXIa, APC, plasmin, single chain t-PA, plasma kallikrein, and C1s have then been related to the reactivity of chromogenic substrate S-2238™ with thrombin, and proved to be 5, 5, 40, 5, 5, 60, and 2%, respectively.

What is the proteolytic mechanism of serine proteases?

The bond-cleaving reaction exerted by a serine protease on its substrate is the result of an interaction between the substrate and the charge relay network of the enzyme. This network, which is present in the active site of all serine proteases, is known as the catalytic triad.

It is built up from the side-chains of three specific amino acids (the hydroxy group of serine, the imidazole group of histidine and the carboxylic acid group of aspartic acid) that interact with each other through an array of hydrogen bonds.

Charge relay network of serine proteases.

The proteolytic action of a serine protease on its substrate comprises several steps starting with the formation of a non-covalent complex between the enzyme and the substrate. A nucleophilic attack by the serine hydroxyl group on the amide carbonyl carbon atom in the substrate results in cleavage of the amide bond and the formation of an acyl-enzyme intermediate.

Formation of an acyl-enzyme intermediate.

The acyl-enzyme ester bond is then hydrolyzed in the rate limiting step and the enzyme is now free to catalyze the cleavage of another substrate molecule.

Hydrolysis of the acyl-enzyme intermediate.

What is a chromogenic substrate?

Enzymes are proteins that catalyze most of the chemical reactions that take place in the body. They make it possible for chemical reactions to occur at neutral pH and body temperature. The chemical compound upon which the enzyme exerts its catalytic activity is called a substrate.

Proteolytic enzymes act on their natural substrates, proteins and peptides by hydrolyzing one or more peptide bond(s).

This process is usually highly specific in the sense that only peptide bonds adjacent to certain amino acids are cleaved.

Chromogenic substrates are peptides that react with proteolytic enzymes under the formation of color. They are made synthetically and are designed to possess a selectivity similar to that of the natural substrate for the enzyme.

Attached to the peptide part of the chromogenic substrate is a chemical group which when released after the enzyme cleavage gives rise to color. The color change can be followed spectrophotometrically and is proportional to the proteolytic activity.

The chromogenic substrate technology was developed in the early 1970s, and has since then become a tool of substantial importance in basic research.

The majority of chromogenic substrate applications are found in various clinical fields. In particular they have been used to generate fundamental knowledge of the mechanisms regulating blood coagulation and fibrinolysis.

Furthermore, products based on chromogenic substrate technology have brought a new generation of diagnostics into the clinical laboratory.

Prothrombin, the natural substrate of Factor Xa, is cleaved by Factor Xa at two positions, each proceeded by the same four amino acid sequence. Factor Xa activity can be determined by the chromogenic substrate S-2222™ which is composed of the same amino acids coupled to a chromophore.

Absorption spectrum of a chromogenic pNA-containing substrate (S) and of pNA. The hydrolysis of a peptide-pNA bond in the chromogenic substrates results in the release of pNA which in turn changes color. Thus the change in absorbance (ΔA/min) is directly proportional to the enzymatic activity. The reaction is usually recorded at 405 nm.