Chromogenix S-2251™ is a chromogenic substrate for plasmin and streptokinase-activated plasminogen. The substrate has been used for the determination of:
- Antiplasmin in plasma
- Plasminogen in plasma
Each vial contains chromogenic substrate S-2251™, 25 mg and mannitol 60 mg as a bulking agent. Stability: Substance: Stable until expiry date if stored at 2-8°C. Avoid exposure to light. The substance is hygroscopic and should be stored dry. Solution: 3 mmol/L in H2O is stable for at least 6 months at 2-8°C. Contamination by microorganisms may cause hydrolysis.
Chemical name: H-D-Valyl-L-leucyl-L-lysinep-Nitroaniline dihydrochloride
Formula: H-D-Val-Leu-Lys-pNA · 2HCl
Mol. wt: 551.6
Plasminogen (MW: 92 Kda) is the zymogen form of plasmin (83 KDa).
The activation of plasminogen is accomplished by t-PA, urokinase or streptokinase.
It is not the purpose of this text to describe the mechanisms of activation of plasminogen and its inhibition, but it is important to underline that they can affect the reaction of the active enzyme with chromogenic substrates, casein or fibrin.
For more information refer to Gaffney (1).
1 ml of normal plasma contains about 180 µg or 2 nmol of plasminogen. The activity of plasminogen can be expressed in casein units (CU) e.g. for example to Sgouris et al. (2).
Friberger (3) found that the amount of plasminogen in 1 ml of normal plasma corresponds to 3.8 CU, as determined by the chromogenic method described (streptokinase activated plasminogen).
The Chromogenix plasminogen reagent when activated with streptokinase shows an activity of 7.3 nkat chromogenic substrate S-2251™ per CU.
In 1983 Friberger (4) reported that 1 µg of plasmin corresponds to 0.20 nkat chromogenic substrate S-2251™, or to 0.024 CU or to 0.028 CTA-U.
In 1975, the first standard for plasmin was established (5) and the international unit was defined equivalent to CTA-U (5-6).
This contained a purified human plasmin in 50% glycerol. When tested by Friberger et al. (7) , it showed about the same activity as the Kabi preparation in terms of µmol of substrate hydrolysed per minute per unit of enzyme. Since then the different aspects of caseinolytic, amidolytic and fibrinolytic activity have been explored in details and, the discrepancy between amidolytic and fibrinolytic activity in reference preparations was underlined as in the case of the 2nd International Reference Preparation of plasmin in 1983 (8).
The current 3rd International Standard for plasmin, 97/536 was established in 1998, and the results presented at the Fibrinolysis Subcommittee of the Scientific and Standardization Committee of the International Society of Thrombosis and Haemostasis (Ljubljana, Slovenia, June 1998). The 2nd International Reference Preparation was used as reference material and a chromogenic assay was the method used. There is no established WHO standard for plasminogen, but the National Institute for Biological Standards and Control, established the Ist British Reference Preparation, 78/646 (9).
It is a preparation of Glu-plasminogen, assayed by fibrinolytic and chromogenic methods against the 2nd International Standard for plasmin. Complete activation of plasminogen was achieved by both urokinase or streptokinase and the activity was comparable to that of the reference preparation of plasmin.
- Gaffney PJ.
Standardization of plasminogen assays. Haemostasis 18 Suppl. 1, 47-60 (1988).
- Sgouris JT et al.
The preparation of human fibrinolysin (plasmin). Vox Sang 5, 357-376 (1960).
- Friberger P and Knos M.
Plasminogen activation in normal plasma. In: Chromogenic peptide substrates, Eds, Scully MF and Kakkar VV, Churchill Livingstone, 128-140 (1979).
- Friberger P et al.
Synthetic peptide substrate assays and fibrinolysis and their application on automates. In: Seminars in Thrombosis and Haemostasis, Vol. 9, 281-300 (1983).
- Kirkwood TB et al.
A standard for human plasmin.
Thromb Diath Haemorrh 34, 20-30 (1975).
- 28th Report, WHO Expert Committee on Biological Standardization. WHO Tech Rep Ser No 610; 1977, pp 13.
- Friberger P et al.
A new specific substrate for the determination of plasmin. In: Chromogenic peptide substrates, Eds, Scully MF and Kakkar VV, Churchill Livingstone, 121-127 (1979).
- Gaffney PJ and Mussett MV.
International collaborative study for the establishment of the second international reference preparation of plasmin. Thromb Haemost 50, 645-649 (1983).
- Gaffney PJ and Curtis AD.
The establishment of a standard for plasminogen. Thromb Haemost 51, 376-378 (1984).
|Chromogenix Chromogenic substrate molecules images|
View our chromogenic substrates
|What happened to the Substrate Toolbox application?|
The Substrate Toolbox app has been discontinued. If you have a specific substrate request, please contact DiaPharma technical support at 1-800-447-3846 or firstname.lastname@example.org.
|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).|
|There are a few different substrates that are hydrolyzed by plasmin. If I want to use as short incubation times as possible, and need a selective substrate for plasmin, which should I choose?|
|The substrates for plasmin include S-2251™, S-2302™, and S-2403™. While S-2251™ is a popular and suitable substrate for detection of plasmin, S-2403™ has a higher kcat/km value. It is a faster substrate, and incubation times can be shorter. S-2403™ is therefore the substrate of choice for this situation.|
|Which substrate is best suited for measuring two-chain tPA, and why?|
|S-2765™, S-2366™, and S-2288™, S-2403™ are suitable for single-chain tPA, and S-2251™ was used in the discontinued Chromogenix Coaset® tPA kit, but there are no substrates specifically for two-chain tPA. A paper by Verheijen et al. (Thromb Res, 1985; 39: 281 – 288), however, describes a method comparing the direct amidolytic activity of tPA on S-2366™ and the plasminogen activating activity. Also, the substrate S-2288™ is suitable for measuring double-chain tPA because it has a slightly higher sensitivity than S-2366™. S-2288™ should be used with purified systems, though, since this substrate is sensitive for several proteases.|
|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 email@example.com.|
|What is a peptide? What is the difference between a tripeptide and a tetrapeptide? How are amino acids linked to form peptides?|
|A peptide is the name assigned to short polymers of amino acids. They are classified by the number of amino acid units in the chain, called amino acid residues. Tripeptides have three amino acid residues while tetrapeptides have four. A polypeptide is formed when the chain of amino acid residues exceeds several dozen in length. A protein is a molecule composed of one or more polypeptide chains.
Proteins are unbranched polymers of amino acids linked head to tail from carboxyl group to amino group, through formation of covalent peptide bonds. The peptide backbone of a protein consists of the repeated sequence.
-N-Ca– C, where N represents the amide nitrogen, Ca represents the a-carbon atom of an amino acid in the polymer chain, and the final C is the carbonyl carbon of the amino acid. This C is in turn linked to the amide N of the next amino acid, and so on down the line. The unbranched polypeptide chain has two ends, an amino-terminal or N-terminal end and a carboxyl-terminal or C-terminal end.
|Are substrates available in alternate sizes such as 5mg, 500mg, 1g, etc?|
|Please inquire at firstname.lastname@example.org.|
|What substrate research methods are available?|
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:
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:
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:
|Protein concentrations in plasma|