Enzyme assay volumes

Babiak P, Reymond JL (2005) A high-throughput, low volume enzyme assay on solid support. Anal Chem 77 373-377... 

In order for the indicator reaction to parallel the test reaction the ratio, Vmax/ATm, for the indicator enzyme should be significantly greater than the same ratio for the test enzyme. The value for Vmax is the only variable in the equation and is determined by the amount of enzyme present, usually quoted in units per assay volume. An increase by a factor of 100 is usually recommended for each stage and will permit concentrations of approximately 1/100 of the Km value to give a rate comparable to the test reaction rate. It may be necessary, if pH or inhibition effects reduce the activity of the enzyme by a known proportion, to increase further the amount of enzyme added to the assay. Table 8.3 lists some examples of coupled and direct assays for a range of enzymes. 

Knowing the total assay volume the actual amount of product can be calculated from the concentration alue. gi ing the value for the numlicr of units of enzyme activity in the assay. 

If 1.0 ml of enzyme preparation in a total assay volume of 5.0 ml results in the formation of a concentration of 0.3 mmol l-1 of product in 10 minutes, what is the activity of the enzyme preparation in katal ml 1 ... 

Interference by other thiols (e.g. as enzyme buffer components) Homogeneous assay, commerically available components Heterogeneous assay, very low assay volume possible Heterogeneous assay... 

Figure 1. Inactivation of lysozyme by ozone. Volumes of 4.8 ml of O.IM buffer containing 2.814 mg of lysozyme were exposed to a gas stream of ozone in oxygen (1.8 nmole/min ozone). Aliquots were removed for enzyme assay as described in Materials and Methods.

The reaction mixture in the enzyme assay contained, in a total volume of 0.02 ml, 0.25 mM M3-octyl (Maria 1,6 1,3Man/3-octyl, synthesised in the laboratory of Prof. H. Paulsen, Hamburg, Germany), 0.1 M MES, pH 6.5, 0.1 M NaCl, 20 mM... 

Besides the enzymatic incubation in the reaction mixture, all procedures are carried out at 4°C. PTPS activity is assayed by measuring the biopterin produced upon enzymatic incubation at 37°C for 120 min in a final volume of 110 pi in the dark (due to the light sensitivity of pterins), followed by chemical oxidation. To stabilize the produced BH4, DHPR and NADH are present in the enzyme assay. Two separate blanks are prepared, a blank reaction with cell lysate that is immediately oxidized to detect the biopterin that was present in the lysate, and a blank reaction without cell lysate to detect the biopterin that is generated from the incubation (substrate) buffer. 

To determine whether increased rates of 02 uptake can be observed due to the presence of DPO in a latent form, repeat steps 6 to 12 using the optimized enzyme concentration and adding 0.1 ml of 1 % SDS to the enzyme assay mixture. Reduce the volume of water added by 0.1 ml to maintain a total volume of 3 ml. 

Assay volumes usually range from 3 pL (for 1536-well MTPs) to 50 pL (384-well MTPs). Within a given total assay volume, smaller volumes of reagents are added. Frequently, we find it convenient to add reagents into the assay in equivalent volumes of assay buffer. As an example, for a 15-pL assay, one might add 5 pL of compound solution, 5 pL of enzyme stock solution, 5 pL of substrate mix, followed by 10 pL of quench solution in a stop buffer. For kinase assays, the stop buffer may be EDTA and for phosphatase assays, sodium orthovanadate. 

A so-called kinetic assay, in which the reaction rate is followed continuously, is advantageous because it is possible to observe directly the linearity or nonlinearity of the response with respect to time. Many enzyme assays, however, are based on a single measurement at a defined time, a so-called fixed-time assay. It is usually not possible to predict the appropriate amount of enzyme in either kinetic or fixed-time assays to obtain an optimum velocity like that of Assay 2 in Figure 11-14. This may be empirically determined by a dilution experiment in two stages. At first, constant volumes of serial 10-fold dilutions of enzyme are assayed to find the range of dilution in which the calculated activity is maximal and constant (see Figure 11-15). 

To illustrate the rapidity of HPLC, particularly in comparison with the more conventional techniques, the same sample was separated by conventional ion-exchange chromatography. Figure 5.10 compares the two procedures. These data show that where 14 hours was required for the traditional method, only about 45 minutes is required with HPLC. Therefore, the total time needed to carry out this purification, not counting the time for the enzyme assay, could be as short as 3 to 4 hours. If necessary, the chromatography step could be completely automated. Finally, since each run will use only a fraction of the total volume of the starting material, the entire procedure will be economical. 

Finally, the pH at which the purification is carried out may not be suitable for the assay. To solve this type of problem, many investigators, particularly those more familiar with fractions containing volumes such as 10 to 20 mL, usually adjust the sample solution to the assay conditions, either by dilution of an aliquot of the fraction into the buffer used for the enzyme assay or by removing 1 to 2 mL from each and, by dialysis, changing the buffers. 

The enzyme assay contained 0.1 mM 5,6-dihydroxyindole-2-carboxylic acid in 30 mM Tris-HCl buffer (pH 7.8), 2.5 mM MgCl2,1 mM 5-adenosylmethio-nine (as methyl donor), 0.5% Triton X-100,0.5 mM EGTA, and 5 mAf dithio-threitol in a total volume of 250 / L. The reaction was stopped with 25 fiL of 4 M perchloric add. Following centrifugation, the supemate was analyzed by HPLC. Product formation was linear with time for 60 minutes. 

The enzyme assay mixture contained in a final volume of 200 //L 20 //mol glycine-KOH buffer (pH 9.5), 0.30 //mol serotonin-HCl, 0.12 //mol acetyl-CoA, and 50 //L of enzyme solution with a maximum activity of 100 mU/mL. After 5 minutes of incubation at 35°C, the reaction was stopped by diluting 10-fold with 0.1 M perchloric acid. After filtering, a 25 //L aliquot of the filtrate was injected onto the HPLC column. 

The enzyme assay contained 5 mAf hippuryl-His-Leu, 100 mAf phosphate buffer (pH 8.3), and 276 mAf NaCl in a final volume of 650 /tL. When rat lung extracts were the enzyme source, the assay mixture was maintained at... 

Enzyme assays were carried out in a total volume of 100 p.L containing 50 mAf Hepes (pH 7.4), 5 to 20 /xL of enzyme, and L-[6-,4C]dihydroorotate. The reaction as continued for 20 to 30 minutes at 37°C, and stopped by addition of 100 /xL of 1% SDS. After a brief incubation, 200 /xL of the HPLC elution buffer was added and the sample was centrifuged before analysis by HPLC. The rate was linear up to 60 minutes when 2.45 /xA/ dihydroorotate was used as substrate and conversion was less than 20%. 

The enzyme assay contained in a total volume of 250 /xL 50 m M Tris-HCl buffer (pH 7.8), 50 mM 2-mercaptoethanol, 5 mil/ formaldehyde, and 1 mM methylenetetrahydrofolate. The reaction was initiated by adding 25 /xL of 1 mM deoxyuridylate. After 30 minutes at 37°C, the reaction was stopped by addition of 250 /xL of ice-cold 1 M perchloric acid. After 30 minutes in an ice bath, the reaction mixture was centrifuged and the resulting supernate was neutralized with 0.1 volume of 10 M KOH containing 1 M KH2PO4. The supernate obtained by centrifugation was injected onto the HPLC column. Formation of deoxythymidylate was linear with time up to 30 minutes, and with protein in the range of 80 to 720 /xg. 

Assay Reagent Volumes for Endotbelin-Converting Enzyme Assay ... 

At the conclusion of dialysis carefully cut off the top of the dialysis bag and transfer its contents to a graduated vessel and note its volume. The preparation may be stored frozen in this condition and is designated as supernatant VI. A 0.5 ml sample should be removed for protein and enzyme assay. 

If the enzymes are delivered in very small volumes compared to the total assay volume, the dilution will be negligible. For example, the assay volume might be 1.0 or 3,0 ml (usual cuvette sizes) while the assay enzymes might be added in lO /rl volumes. 

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