|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
Will elevated levels of plasminogen interfere with the Chromogenix Coamatic® Plasmin Inhibitor test?
No, elevated plasminogen levels should not effect the results. Residual plasmin hydrolyzes the chromogenic substrate, but the principle of plasminogen is different. No part is played by plasmin. Instead, determination by chromogenic substrate requires all plasminogen in the sample to be activated to plasminogen/streptokinase complex, which hydrolyzes the chromogenic substrate.
Describe the measurement principle behind the discontinued Chromogenix Coamatic® Plasmin Inhibitor kit.
Incubation of diluted plasma with an excess of plasmin results in a rapid complex formation between functional plasmin inhibitor present in the plasma and plasmin. The inhibited plasmin activity is proportional to the amount of plasmin inhibitor. The remaining amount of plasmin hydrolyzes the chromogenic substrate S-2403™, thus liberating the chromophoric group pNA.
In the Chromogenix Coamatic® Protein C assay, what substances could interfere with the assay, how will they affect results, and what can be done to overcome the interference?
A low protein C activity is expected in aprotinin treated patients because aprotinin is an inhibitor of activated protein C. Oral anticoagulant therapy interferes with the formation of g-carboxyglutamic acid moiety of the protein C molecules during biosynthesis in the liver, which results in a loss of anticoagulant activity. Non-carboxylated forms of protein C molecules that are inactive in vivo can still be activated by snake venom or thrombin-thrombomodulin and retain amidolytic activity in vitro. Assays using chromogenic substrates will therefore over-estimate the true level of protein C activity in plasma from patients receiving OAC’s. Streptokinase also influences the hydrolysis of S-2366™. Sample blank activities should be determined with plasma from patients with thrombolytic disorder treated with streptokinase, as well as plasma where contact activation is suspected, should be compared to sample blank activities. A high blank activity may indicate contact activation has occurred. S-2366™ is also sensitive to thrombin. This interference can be quenched by the addition of a thrombin inhibitor such as I-2581.
During the last years recent publication have been showing that the APC Resistance phenotype is a risk factor for venous thrombosis, irrespective of the FV Leiden mutation, and hence this illustrates that the Coatest® APC Resistance and the Coatest® APC Resistance V kits measure different entities. For this reason, both Coatest® APC Resistance and the Coatest® APC Resistance V kits are included in the thrombophilia screening panel in some important laboratories. A recent and interesting publication on inherited thrombophilia was done by Uri Seligsohn and Aharon Lubetsky. In this paper, both APCR and APCR V are suggested as part of six high priority tests. The diagnosis for factor V Leiden should be confirmed by genetic test, in order to decide whether the family members should be examined.
Variations in plasma levels of protein C have no influence on the APC ratio since a standardized amount of exogenous APC is added.
A study by Colucci et al. (Thromb. Haemost. 1994, 72:987 – 988) using the classical APTT method (no FV-deficient plasma pre-dilution) showed that changes in FV levels between 12.5-100% did not modify the response to APC. Other experience has shown that the Coatest® APC Resistance V test may provide ratio values approximately 0.3 units below the median level, which can be fairly close to the cut-off value, when FV levels are 0-40%. This may be explained by the fact that although there is often no abnormal bleeding tendency in heterzygotes with factor V deficiency, prolonged PT and APTT times are observed (ref: Sartori et al. Familial association of hypoplasminogenemia and heterozygous factor V deficiency. Clin Appl Thromb Hemost. 1999; 5(4): 277-281; Salooja N et al. Severe factor V deficiency and neonatal intracranial haemorrhage: a case report. Haemophilia. 2000; 6(1): 44-46. One might also see a slightly decreased ratio in patients with severe liver disease.
According to Chromogenix, FVIII samples above 1.8 IU/ml may result in a reduction of the APC ratio of approximately 0.2 units, although the actual correlation between FVIII activity and APC ratio appears to be weak. When sampling, therefore, the patient should be at rest in order to decrease the FVIII level due to stress.
Can I use Chromogenix Coatest® APC Resistance V without the predilution with FV deficient plasma? Isn’t this just like using the APC R kit?
No. There is more APC in the APC V kit than in the classic kit. This makes the APC ratios twice as high among normals when not using the V def. plasma as a pre-diluent compared to the classic method. Furthermore, using the APC r-V without the pre-diluent will cause a constant change of the cut-off value due to batch-to-batch variations. This effect is not there if you use the V def. plasma, since it normalizes the plasma and ensures that the cut-off value stays the same from one batch to another.
Can I freeze the reagents in the Chromogenix Coatest® APC Resistance kits to prolong their stability?
The APTT reagent cannot be frozen. The other reagents can, but should be rapidly thawed at 37°C and cannot be re-frozen.
Hirudin is the active anticoagulant obtained from the leech, and is now produced by recombinant technology. It is the most potent and specific known inhibitor of thrombin. It inactivates thrombin by blocking the substrate binding groups following the formation of a 1:1 stoichiometric complex. All proteolytic functions of the enzyme are blocked, as is the activation of factors V, VIII, XIII, and the binding of thrombin to platelets. Heparin works indirectly, requires antithrombin as a cofactor, is not effective against thrombin that is already bound to the fibrin clot, and can be inactivated by PF4 or plasma proteins. Heparin therapy also leads to HIT in 5-15% of patients. Conversely, hirudin is the prototypical direct inhibitor of thrombin. It is a more potent anticoagulant and affects clot-bound thrombin, does not require any cofactors, and is not inactivated by PF4 or plasma proteins. Research has suggested that hirudin my provide a small advantage over heparin in situations such as acute coronary syndromes, but cost-benefit analyses are still needed. Hirudin levels can be determined chromogenically using substrate S-2366™ or S-2238™.
The dilution of the 0.1 IU/ml heparin solution in Chromogenix Coatest® Heparin can cause some confusion. Please explain the Coatest® Heparin standardization.
Keep in mind the final concentration of heparin is 0.1 IU/ml plasma. Following the package insert, there is a known concentration of 0.1 IU/ml heparin in the standard, regardless of the dilution, provided the samples are all treated the same way. 100 ml of the 0.1 IU/ml heparin dilution is equivalent to a heparin concentration of 0.1 IU/ml plasma in the assay. This concentration should be compared to the patient-sample. If the analyst takes 100 ml of the heparin dilution (0.1 IU/ml) and dilutes it 1:10, it is comparable to 100 ml of the patient plasma also diluted in the same way. If the analyst get the same absorbance for the sample as for the standard, it contains the same amount of heparin. The important thing is the analyst must always treat patient samples in the same manner as the standards.
In the Chromogenix Coatest® Heparin package insert, the section Limitations of the procedure states that in some pathological states, plasma alone my hydrolyze S-2222™, and that to determine the interference one should substitute FXa with an equal volume of buffer…Why is this necessary?
In some clinical situations such as sepsis, DIC, and cancer, the plasma itself could contain enzymes that might be able to hydrolyze S-2222™. If that happened, background activity could be seen and would therefore lead to an underestimation of the heparin level in the sample. To rule out background activity in the sample, the assay can be run without FXa and instead, and equal volume of buffer is substituted. The absorbance obtained is then subtracted from the absorbance from the normal run.
I need an assay that complies with the USP monograph for the determination of heparin activity. What test kits are suitable?
The USP states that the activity of heparin sodium and heparin calcium should be determined by both a clotting assay and a chromogenic assay. The chromogenic assay consists essentially in the measurement of the anti-FXa activity of the test preparation against the USP Heparin Sodium Reference Standard. All of the chromogenix heparin kits meet this specification. Antithrombin, FXa, and the chromogenic substrates from Chromogenix are suitable for the USP guidelines. The anti-FXa assays are more specific since they measure the ability of heparin-accelerated antithrombin to inhibit a single enzyme. Either plasma or purified AT can be used. More precise determination of unfractionated heparin and low molecular weight heparin are possible.
What is the difference between Chromogenix Coamatic® Heparin, Coatest® Heparin and the discontinued Coatest® Low Molecular Weight Heparin / Heparin?
All of our heparin kits are for the chromogenic determination of UF and LMW heparin in human plasma, and measure the ability of heparin to catalyze the inhibition of FXa by antithrombin.
Coamatic® Heparin is a one-stage assay optimized for a wide range of instruments that does not require the addition ofexogenous antithrombin. It features the use of substrate S2732™, simple reagent preparation, few components, and a simple, straight-forward assay procedure and is usually performed with undiluted plasma.
Coatest® Low Molecular Weight Heparin was mainly intended for the non-automated lab and allows rapid and reliable manual determination in a one-stage procedure. Like Coamatic® Heparin, it used S-2732™, and although slightly dependent on the sample’s AT concentration, no exogenous AT is added.
All of the heparin kits from DiaPharma can. Obviously, when measuring LMW heparin samples, LMW heparin standard, like Fragmin, must be used unless a specific universal calibrator is used. DiaPharma currently has calibrators available for LMW heparin and UF heparin.
Add to the assay an equal volume of 1 IU/ml AT as the plasma volume. AT, 10 IU, can be bought from DiaPharma. Whenever a sample is tested with exogenous AT, it should be measured against a standard curve also run with exogenous AT.
Chromogenix Coamatic® Heparin is optimized for use with and without exogenous antithrombin. When is it recommended to add exogenous AT, and why?
It is recommended to add exogenous antithrombin when children below the age of one year are being tested. Although exogenous AT has been shown to be needless for patients with AT levels between 35-135%, pre-term newborns can have levels as low as 30%. Sufficient studies of Coamatic® Heparin have not been performed on infants, so as a precaution exogenous AT should be added. Also, for measuring heparin activities in serum, AT is needed since endogenous AT activity will be very low.
Yes, it will detect FVIII activity in dog, cat, horse, rabbit, and mouse plasma.
We know that low levels of factor VIII activity constitutes hemophilia A, but are there any clinical manifestations of elevated factor VIII levels? How can a researcher measure elevated FVIII levels?
There is evidence that a high level of factor VIII is a risk factor for venous thrombosis. There are two articles in Thrombosis and Haemostasis 2000; volume 83:
1.Kraaijenhagen et al. High plasma concentration of factor VIII:c is a major risk factor for venous thromboembolism (p. 5-9)
2.O’Donnell et al. Elevation of FVIII:c in venous thromboembolism is persistent and independent of the acute phase response (p. 10-13)
Both of these recent articles discuss the issue of elevated factor VIII levels as a risk factor for venous thrombosis.
An adaptation of Chromogenix Coamatic® FVIII has been developed to allow accurate determination of elevated factor VIII levels. The method is for research use only.
Sometimes when I run the Chromogenix Coamatic® FVIII assay, I see an upward drift in my activity from the first to the last samples. Why is this?
Thrombin, 1 NIH unit, is included in the factor reagent. It has been shown that thrombin can activate FX, and over time you will see an increase of up to 5% activity. If you are doing numerous samples at once on an automated instrument, you might want to consider Coatest® FVIII.
No, glass surfaces may interfere with the generation of FXa. Use plastic tubes for the manual method.
If thrombin is formed, will it hydrolyze the substrate S-2765™ when using Chromogenix Coamatic®, Coatest® SP, or Coatest® SP4 FVIII kits?
No, hydrolysis of S-2765™ by thrombin is prevented by the addition of the synthetic thrombin inhibitor I-2581.
The standard pre-dilutions for the Coatest® SP FVIII assay dont make sense to me. For example, to get 100%, I dilute 100 ml plasma with 50 ml buffer, but to get my 50% standard, I dilute 100 ml plasma with 200 ml buffer. Why?
The key is that the plasma that does not have a pre-dilution corresponds to 150%, not 100%. One way to look at it is that since 100 is 67% of 150, then the plasma must be diluted 67%, which corresponds to 100 ml plasma with 50 ml buffer (100 plasma / 150 total volume = 0.67). This process can be followed for all of the standard pre-dilutions.
Clinicians or researchers interested in determining FVIII levels in patients with hemophilia A or with elevated factor VIII levels, which may be associated with thrombotic risk. Also, manufacturers interested in determining factor VIII potencies. The chromogenic FVIII activity method has been selected as the reference method by the EP for potency estimation of VIII concentrates, and it is a valuable tool for diagnosis an monitoring of hemophilia and for thrombophilia screening.
While clotting assays are cheap, rapid, and simple to perform, they are sensitive to pre-activation, show interferences in the assays, and can give overestimation of FVIII concentrates. They also require a considerable amount of FVIII deficient plasma.
Coamatic® FVIII is also rapid and simple, and has additional benefits. It is suited for a wide range of automated instrument applications, there is no heparin influence, and it is highly sensitive, precise and accurate. The low measurement range provides a reliable tool for the classification of bleeders.
What is the difference between Chromogenix Coamatic® FVIII, Coatest® SP FVIII, and Coatest® SP FVIII?
All of these kits measure the same thing — factor VIII activity in plasma and potency estimation of FVIII concentrates. Coamatic® FVIII includes the FXa substrate S-2765™, and the factor reagent FIXa + FX and thrombin colyophilized with CaCl2 and phospholipid. The Coatest® SP FVIII includes the FXa substrate S-2765™, and the FIXa + FX reagent, CaCl2, and phospholipid are all separate components in the kit. Incubation times are a little longer, and the reagent prep is less simple. Coatest® SP4 FVIII is exactly the same as Coatest® SP FVIII, but the FIXa + FX reagent is divided into 4 vials of 1.8 U (reconstituted with 3 ml) instead of 1 vial of 6 U (reconstituted with 10 ml).
First, factor VIII circulates in the plasma bound to von Willebrand factor (vWf). Thrombin cleaves and activates factor VIII and releases vWf. The vWf is then free to bind to ruptured endothelial cell surfaces where it activates platelet aggregation. The released FVIIIa acts as a cofactor of factor IXa to generate factor Xa. In the presence of Ca2+ and phospholipids, FX is activated to FXa by FIXa (look at the coagulation cascade for a better understanding). Since FVIIIa is a cofactor to FIXa, it greatly stimulates the reaction. By using optimal amounts of Ca2+, phospholipid, and FIXa, and an excess of FX, the rate of activation of FX is linearly related to the amount of FVIII. FXa hydrolyses the chromogenic substrate S-2765™ which releases the chromophoric group pNA. The color is read at 405 nm, and generated FXa and thus the intensity of color, is proportional to the FVIII activity in the sample.
Thrombin activates FX, and this happens when the reagent is reconstituted. An incubation step is required in the Chromogenix Coatest® FVIII kit.
Yes. Predilute cryoprecipitate in the Coatest® FVIII kit buffer to 1 IU/ml, then follow the kit insert. Somewhat larger dilutions than described in the kit may be necessary with some concentrations.
Even though there is documentation that the anti-FXa method is better, how could a researcher determine antithrombin in plasma based on a thrombin method?
A thrombin-based chromogenic heparin cofactor assay for the determination of antithrombin activity can be performed using Chromogenix S-2238™ as described on the Antithrombin Method tab of the Chromogenix S-2238™ product page.
Studies have shown that thrombin-based AT assays, such as Coatest® AT, show an overestimation of AT activity in patients on heparin therapy due to the influence of heparin cofactor II. The FXa-based method provides more valid results in patients on heparin therapy. There is no influence from heparin cofactor II, a2-macroglobulin, or a2-antitrypsin. The AT FXa assay is a better discriminant between AT deficient and non-AT deficient individuals than the thrombin based assay.
In plasma where contact activation has occurred, a contribution to the substrate activity might be produced. An underestimation of AT level may follow. A blank can therefore be performed, and the value obtained in the absence of FXa can be subtracted from the sample value.
How should I make my standard pre-dilutions? It is not very clear from the Chromogenix Coamatic® Antithrombin package insert.
A suggested method for the predilution, although it cab be up to the analyst what volumes to use:
100%: 400 ul normal plasma, 0 ul Saline
75%: 300 ul normal plasma, 100 ul Saline
50%: 200 ul normal plasma, 200 ul Saline
25%: 100 ul normal plasma, 300 ul Saline
From here, follow the package insert.
The Chromogenix Factor Xa reagent says it is 71 nkat, but I need to know what that is in g/ml and mol/ml.
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. The conversion is as follows:
FXa has a MW approximately 44,000.
The specific activity for FXa is 1.9 nkat/mg, as determined with chromogenic substrate S-2222™.
This gives that 71 nkat corresponds to 37.4 mg FXa (=37.4 x10-6 g FXa)
This corresponds to 8.5 x 10-10 mol = 0.85nmol.
The concentration in mol/l and g/l will depend on the dilution volume you choose.
Note: 1 IU corresponds to 20 nkats, which means 71 nkatS-2222 = 3.55 IU.
S-2337™ was previously included in the now discontinued Chromogenix Coatest® FX kit, so another substrate must be employed. S-2765™, which is also a substrate for FXa, can therefore be used, and a method based on the activation of FX in the presence of calcium using Russell’s Viper Venom (RVV) as the activator is described in the Chromogenix catalog. DiaPharma has composed a new DiaPharma Factor X kit based on S-2765™.
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.
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.
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.
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.
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.
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).
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.
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).
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.
Please inquire at firstname.lastname@example.org.
- 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.
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:
- 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.
- 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:
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:
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The plasminogen present in the sample is activated by the addition of an excess of streptokinase (Sk) forming a plasminogen-streptokinase (Plg/Sk) complex. Plg-depleted fibrinogen is included in the Sk reagent in order to avoid the risk of overestimation of Plg in the pathological plasmas containing elevated levels of fibrinogen (Fib) and/or fibrin degradation products (FDP). The PlgSk/Fib complex is determined by the rate of hydrolysis of the chromogenic substrate S-2403™. The pNA release measured at 405 nm is proportional to the plasminogen activity level of the plasma sample.
5 IU corresponds to 1 mg. Therefore a 25 IU vial corresponds to 5 mg AT. The molecular weight of AT is 58 kDa.