Chromogenic Substrates

 

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CHROMOGENIC SUBSTRATES UNIVERSITY | CHROMOGENIC SUBSTRATE FAQ


What is a chromogenic Substrate?

Proteolytic Enzymes

Serine Proteases

Proteolytic Mechanism

Specificity and Selectivity

Enzyme Kinetics

Substrates in Practice

Selectivity Tables

Kinetic Tables

Protein Concentrations in Plasma

Theoretical Basis for Calculation

International Units and Enzyme Activity

FAQ

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chromogenic substrate questions

· What is a peptide? What is the difference between a tripeptide and a tetrapeptide? How are amino acids linked to form peptides?

· Define an enzyme and a substrate.

· What is a chromogenic substrate composed of?

· Define a katal.

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

· Why are the reactions measured at 405 nm?

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

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

· Which substrate is best suited for measuring two-chain tPA, and why?

· 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 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?

· If I want to determine FX in plasma, but do not want to buy a kit, what other options are there?

· The factor Xa reagent says it is 71 nkat, but I need to know what that is in g/ml and mol/ml.

· What is the 53 nkat vial of thrombin equivalent to in NIH units?

· The activity of plasmin and plasminogen is expressed in CU. What is this, and what is the equivalent in nkat?

· Is the plasminogen reagent Glu- or Lys- plasminogen?

· What constitutes the tPA stimulator, and how is it prepared?

· Can the tPA activity standard be used to measure two-chain tPA or just single chain tPA?

· The tPA standard dilution of 10 IU/ml is confusing in the Coaset tPA kit. Can you explain it?

· I am looking for a chromogenic assay to measure TFPI activity. What substrates will work?

· How can I measure prothrombin activity? What is considered an elevated level of prothrombin activity?


chromogenic substrate answers

· 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.

· 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.

· 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 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 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).

· Why are the reactions measured at 405 nm?

chromogenic substrate FAQ

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.

· 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.

· 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.

· 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 is used in Coaset tPA, 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.

· 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, S-2390 (discontinued in the 25 mg quantities), 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.

· 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).

· If I want to determine FX in plasma, but do not want to buy a kit, what other options are there?

Since S-2337, which is included in the Coatest FX kit, is not available for separate purchase, 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 for research use only based on S-2765.

· The 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.

· What is the 53 nkat vial of thrombin equivalent to in NIH units?

Based on an assay with S-2238, for bovine thrombin,1 NIH-U = 1.15 IU = 3.4 nkat. According to Chromogenix, bovine thrombin, 53 nkatS-2238 is equivalent to approximately 21 NIH-U or 25 IU.

· The activity of plasmin and plasminogen is expressed in CU. What is this, and what is the equivalent in nkat?

CU stands for Casein Units, and is a measure of the proteolytic activity on the substrate casein. The plasminogen reagent when activated to plasmin with streptokinase shows and activity of 7.3 nkatS-2251 per CU. 1 mg of plasmin corresponds to 0.20 nkatS-2251, or to 0.024 CU.

· Is the plasminogen reagent Glu- or Lys- plasminogen?

It is a preparation of at least 95% Glu-plasminogen.

· What constitutes the tPA stimulator, and how is it prepared?

The tPA stimulator is a lyophilized powder of 3 g CNBr-digested human fibrin(ogen) fragments prepared by the degradation of fibrinogen with cyanogenbromide. These fragments have excellent stimulating properties, are easily soluble, and can be prepared simply, quickly, and reproducibly.

· Can the tPA activity standard be used to measure two-chain tPA or just single chain tPA?

The t-PA 10 mg activity standard is composed 95% of single-chain t-PA with a specific fibrinolytic activity of about 500,000 IU/mg and is not suitable to measure the two chain t-PA, even they have different activity on synthetic peptide substrates.

· The tPA standard dilution of 10 IU/ml is confusing in the Coaset tPA kit. Can you explain it?

First, assume a patient sample contains 10 IU/ml plasma. When the blood has a hematocrit of about 50%, the plasma volume will constitute about 50% in the sample. All tPA is present in the plasma fraction. The initial collection of blood in citrate reduces the tPA plasma concentration about 10%, resulting in a concentration of about 9 IU/ml.  Mixing 1 volume of citrated blood with 1 volume of acetate buffer corresponds, with a hematocrit of 50%, to mixing 0.5 volumes of plasma with 1 volume of acetate buffer. The plasma is diluted 1:2, and at this stage has a tPA concentration of 3 IU/ml. The addition of 10 ml HCl to 150 ml acidified plasma results in a tPA concentration of 2.8 IU/ml. Thus, a plasma sample containing 10 IU/ml will actually contain 2.8 IU/ml, after the prescribed procedure. The 10 IU/ml tPA standard is diluted from 20 ml to 340 ml (100 ml tPA/PAI depleted plasma + 200 ml acetate buffer + 20 ml HCl + 20 ml tPA standard) and its actual concentration is therefore 50 x 20/340 = 2.9 IU/ml.

· I am looking for a chromogenic assay to measure TFPI activity. What substrates will work?

TFPI is a direct inhibitor of FXa, and the TFPI/FXa complex inhibits TF/FVIIa. The chromogenic assay for TFPI is based on the ability of TFPI to inhibit TF/FVIIa catalytic activity in a reaction mixture that contains diluted plasma and known amounts of TF, FVIIa, and FXa. Contact DiaPharma for a detailed protocol.

· How can I measure prothrombin activity? What is considered an elevated level of prothrombin activity?

A mutation G ® A in the untranslated 3’ region of the prothrombin gene at nucleotide position 20210 (G20210A mutation) constitutes a risk factor  for venous thromboembolism. About 90% of carriers of the mutation have elevated levels of prothrombin activity. The normal range for non-carriers is about 75 – 130%. There is a research method available to measure prothrombin activity levels up to 175% using the thrombin substrate S-2238. It is not specific for the G20210A mutation.


Note: The Substrate catalog is a good source of basic and advanced information about chromogenic substrates.

 

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