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Light Scattering Enzyme Assays

Creative Enzymes is a major player in the global market of enzyme services. We are specialized in activity measurement and kinetic assay, especially using light scattering assays. As a well-known service provider, Creative Enzymes is recognized as a leader in the development of unique and unrivaled bioanalytical services for various enzymes serving the needs of the pharmaceutical, biotechnological, and diagnostic industry. Through most up-to-date facilities and industry-leading technologies, we provide innovative bespoke enzyme services to customers.  

Enzymes are widely used in both large-scale industrial and precise biotechnical processes. Good designs and operation of these process rely on high efficiency and long-term stability of the enzyme, which are affected by many parameters such as the temperature, ionic concentration, and pH. Therefore, it is important to be able to constantly test and monitor enzyme activities in the early development stage and through the whole production process. Light scattering is a frequently used assay to determine enzyme activity and contents, showing the advantages of high sensitivity and signal-to-noise ratios. Different from absorbance spectroscopy, light scattering is a form of scattering in which light in the form of propagating energy is scattered. Light scattering can be considered as the deflection of a ray from a straight path, for instance, by irregularities in the propagation medium, particles, or in the interface between two media. When these irregularities are considered to be random and dense enough that their individual effects average out, this kind of scattered reflection is commonly referred to as diffuse reflection (Figure 1).

Mechanisms of diffuse reflection include surface scattering from roughness and subsurface scattering from internal irregularities such as grain boundaries in polycrystalline solids. Figure 1: Mechanisms of diffuse reflection include surface scattering from roughness and subsurface scattering from internal irregularities such as grain boundaries in polycrystalline solids.

A common dichotomy in light scattering terminology is static light scattering (SLS) versus dynamic light scattering (DLS). In SLS, the experimental variable is the time-average intensity of scattered light, whereas it is the fluctuations in light intensity in DLS that are researched. Nowadays, both techniques are used for the assay of enzymes. The assay uses substrate-coated colloidal particles. The general methodology takes advantage of the fact that unstabilized particles tend to aggregate. Hydrolysis of the substrate coat on the particles causes the particles to become unstable and therefore aggregate. The aggregation rate is proportional to the enzyme concentration. The initial disaggregation reaction is the important parameter, which is monitored by light scattering. The aggregation of the particles can also be measured by dynamic scattering. Although turbidimetric assays follow the same general principle as light scattering assays, turbidimetry is of significant lower sensitivity and reproducibility.

DLS, also known as quasielastic light scattering and photon correlation spectroscopy. It has been applied in size and shape analyses of macromolecules and used to study various inter- and intramolecular interactions, including association, aggregation, gelation, micellization, and molecular conformation, and the effects of many different factors on molecular structure. Dynamic light-scattering methodology measures a photon autocorrelation function that is initially characterized by a single decay constant whose value is related to the single particle diffusion coefficient. As soon as the particles lose a portion of their substrate coat, they start to aggregate, and the substrate autocorrelation function shows an additional component that has a long time-decay constant. The amount of the component with the long decay (slow) relates to the amount of enzyme introduced into the solution. In the case of the enzymes tested, the approximate limit of sensitivity appears to be in the microgram range.

Static light scattering measures the product of weight-averaged molar mass and concentration of macromolecules in solution. Given a fixed total concentration of one or more species over the measurement time, the scattering signal is a direct measure of the weight-averaged molar mass of the solution, which will vary as complexes form or dissociate. Hence the measurement quantifies the stoichiometry of the complexes as well as kinetics.

Hypothetical dynamic light scattering of two samples: Larger particles on the top and smaller particles on the bottom. Figure 2: Hypothetical dynamic light scattering of two samples: Larger particles on the top and smaller particles on the bottom.

Creative Enzymes is one of the few companies that specifically developed and perform in-house light scattering enzyme assays. Having tested a wide range of enzymes, Creative Enzymes has accumulated extensive experiences and is able to provide rapid and superb enzyme activity assays to support all types of research. We are always engaged in providing customer-oriented solutions to help researchers with screening, development, and production of enzyme products and enzymatic processes.


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