Enzyme Mass Concentration

A number of immunoassays for human enzymes and isoenzymes measuring protein mass instead of catalytic activity 

---

Concentration of A Arrhenius constants Arrhenius constant Constant in equation 5.82 Surface area per unit volume Parameter in equation 5.218 Cross-sectional area Concentration of B Stoichiometric constants Parameter in equation 5.218 Concentration of gas in liquid phase Saturation concentration of gas in liquid Concentration of G-mass Concentration of D-mass Dilution rate DamkOhler number Critical dilution rate for wash-out Effective diffusion coefficient Dilution rate for maximum biomass production Dilution rate for CSTF 1 Dilution rate for CSTF 2 Activation energy Enzyme concentration Concentration of active enzyme Active enzyme concentration at time t Initial active enzyme concentration Concentration of inactive enzyme Total enzyme concentration Concentration of enzyme-substrate complex with substance A... 

Photometric measurements performed in clinical laboratories use advanced chemical and biochemical methods and diverse instrumentation. Most analyses performed in clinical laboratories are based on spectrophotometric methods using photometric systems, such as absorption photometers, atomic absorption spectrophotometers and flame photometers. Typically, the result is expressed as mass concentration of analyte in solution (mg/dl), molar concentration (mmol/1), or catalytic concentration of enzyme activities in solution (U/l) [6],... 

CK-MB can be measured in numerous ways. Immunoassays developed in recent years have improved on the analytical and clinical sensitivity and specificity of the earlier immunoinhibition and immunoprecipitation assays. These assays now (1) measure CK-MB directly and provide mass measurements, (2) are easily automated, and (3) provide rapid results (<30 minutes). Mass assays reliably measure low CK-MB concentrations in both samples with low total enzyme activity (<100 U/L) and with high total enzyme activity (>10,000 U/L). Furthermore, no interferences from other proteins have been documented. The majority of commercially available immunoassays that use monoclonal anti-CK-MB antibodies are the same as those listed in Table 5-2 for cardiac troponin assays. Excellent concordance has been shown between mass concentration and activity assays. A primary reference material is commercially available to assist in harmonization. If used for assay standardization, then this material allows... 

As can be seen from Table 8.7 productivity (expressed in g h b is highest for precursor addition. The production of L-phenylalanine from phenylpyruvic add also has the shortest reaction time to obtain hi conversions. The pH commonly used is around 75, quite normal for biological processes. Only the enzyme phenylalanine ammonia lyase shows an optimiim pH of lO.The process temperature varies between 30 and 40°C with an average of 35°C. No extreme temperatures have been reported due to the fact that denaturation occurs at hi temperatures. The optimal concentration for cells frequently used is 10-20 g 1". However, conversion of ACA is done with hi cell mass concentrations in recent studies possibly to compensate for substrate inhibition and thus to maintain hi product concentration. The processes using PPA and ACA need an amino add as amino donor, usually L-aspartic add is used. 

Mati ix AH components of a material system, except the analyte. Measurand The quantity that is actually measured (e.g., the concentration of the analyte). For example, if the analyte is glucose, the measurand is the concentration of glucose. For an enzyme, the measurand may be the enzyme activity or the mass concentration of enzyme. 

Isoenzymes and multiple forms of enzymes may provide additional organ specificity. Table 23-6 summarizes various enzymes, their associated types of malignancy, and the assays used to measure their activity (Act) or their mass concentration (RIA or immunometric assay). Enzymes are traditionally measured by their activities. With the introduction of antibody techniques, some enzymes, such as PSA, are measured as protein antigens rather than by their enzyme activity. 

When unstirred batch membrane units are used as reacting vessels, a steady state mass balance on retained species, i.e., enzymes, leads to the evaluation of their concentration as a function of the distance from the membrane surface, x. Taking into account both convective and diffusive mass transfer mechanisms, the enzyme mass balance equation at steady state is as follows ... 

In this study, we will explore whether an objective function, relating enzyme activity to cost, can be developed to establish the cost function of cellulase and other enzymes. We will determine the cost increase in cmde cellulase (in terms of activity per mass) after processing used to enhance the purity and concentration of this protein. We shall start with a more generalized objective function comprised of measurable purification process responses to create our cost model and reduce that model to the specific cost function for this study. A previously developed objective function quantifies the tradeoff between maximizing the enzyme concentration in a separation process such as a foam fiactionation process and minimizing the loss of enzyme mass and enzyme activity in that process [1] is shown below. 

Enzymes may be measured by monitoring a reduction in substrate concentration or an increase of reaction product. The majority of the common enzymes can be measured using a kinetic approach in which the velocity of the enzyme reaction is monitored by serial measurements over a short time period. For some enzymes, the measurement reactions are linked to the enzyme cofactors nicotinamide adenine dinucleotide (NAD) or nicotinamide dinucleotide phosphate (NADP), and changes in these cofactors are measured in the ultraviolet spectrum. Reactions can be linked through second- or third-step reactions where these cofactors are involved (e.g., for AST and ALT). Other enzymes may be measured colorimetrically (e.g., ALP and GGT). Very few enzymes have been measured as enzyme mass, although proteomic methods may lead to greater utilization of enzyme mass measurements. 

Zhang, Y. and Lynd, L.R. (2003) Quantification of cell and cellulase mass concentrations during anaerobic cellulose fermentation development of an enzyme-linked immunosorbent assay-based method with application to Clostridium thermocellum batch cultures. Anal. Chem., 75 (2), 219—227. 

An expression for the concentration of the El complex can be obtained from the enzyme mass balance and dissociation constants ... 

If the amount of denatured enzyme is being monitored as a function of time instead, the first-order ordinary differential equation that characterizes the increase in the concentration of denatured enzyme and enzyme mass balance are... 

Then, the pH value at which precipitation PE-P is higher should be selected, and a turbidimetric titration curve at a constant enzyme amount with increasing PE concentration is carried out (Fig. 14 (right)). The stoichiometric ratio mass of enzyme /mass of PE can be calculated from the non-lineal fitting of the experimental data. This parameter is important because it allows us to know the minimal amount of PE necessary to precipitate the protein. Table 4 shows the stoichiometry data obtained for different systems previously studied [8, 22, 38, 50]. 

The response of the immobilized enzyme electrode can be made independent of the enzyme concentration by using a large excess of enzyme at the electrode surface. The electrode response is limited by the mass transport of the substrate. Using an excess of enzyme often results in longer electrode lifetimes, increased linear range, reduced susceptibiUty to pH, temperature, and interfering species (58,59). At low enzyme concentrations the electrode response is governed by the kinetics of the enzyme reaction. 

Hundreds of metabohc reac tions take place simultaneously in cells. There are branched and parallel pathways, and a single biochemical may participate in sever distinct reactions. Through mass action, concentration changes caused by one reac tion may effect the kinetics and equilibrium concentrations of another. In order to prevent accumulation of too much of a biochemical, the product or an intermediate in the pathway may slow the production of an enzyme or may inhibit the ac tivation of enzymes regulating the pathway. This is termed feedback control and is shown in Fig. 24-1. More complicated examples are known where two biochemicals ac t in concert to inhibit an enzyme. As accumulation of excessive amounts of a certain biochemical may be the key to economic success, creating mutant cultures with defective metabolic controls has great value to the produc tion of a given produc t. 

Enzymes are excellent catalysts for two reasons great specificity and high turnover rates. With but few exceptions, all reac tions in biological systems are catalyzed by enzymes, and each enzyme usually catalyzes only one reaction. For most of the important enzymes and other proteins, the amino-acid sequences and three-dimensional structures have been determined. When the molecular struc ture of an enzyme is known, a precise molecular weight could be used to state concentration in molar units. However, the amount is usually expressed in terms of catalytic activity because some of the enzyme may be denatured or otherwise inactive. An international unit (lU) of an enzyme is defined as the amount capable of producing one micromole of its reaction product in one minute under its optimal (or some defined) reaction conditions. Specific activity, the activity per unit mass, is an index of enzyme purity. 

---