Molecular weight determination and Characterization of Enzymes

This presentation shows about how the determination of molecular weight is done and what are the different ways or method to determine the molecular weight. This presentation also tells about the enzymes and its characterization.

1. SEMINAR Biochemical techniques and enzymology

2. MOLECULAR WEIGHT DETERMINATION AND CHARACTERISATION OF ENZYMES Prepared by:- AYUSHI SOMVANSHI BT190203 MSc Biotechnology 2 nd Semester

3. enzymes Basic Definition : Enzymes are catalysts that increase the rate of a chemical reaction without being changed themselves in the process. Enzyme activity can be regulated, varying in response to the concentration of substrates or other molecules. They function under strict conditions of temperature and pH in the body. Enzymes are proteins that act as biological catalysts. Catalysts accelerate chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products

4. Some basic properties of enzymes Catalytic Property. Specificity. Reversibility. Sensitiveness to Heat and Temperature. Specific to Hydrogen Ion Concentration (pH)

5. molecular WEIGHT Determination OF ENZYMES When an enzyme has been purified, its molecular weight, more correctly termed relative molecular mass, Mr , may be determined. It can be done by 3 ways:- Gel electrophoresis (SDS-PAGE) Chromatography (size exclusion chromatography and gel filtration chromatography) Spectroscopy (Mass Spectroscopy)

6. GEL ELECTROPHRESIS (SDS-PAGE) Most popular method of determining Mr of an enzyme after purification is PAGE (Poly Acrylamide Gel Electrophoresis) , in the presence of SDS (Sodium Dodecyl Sulphate) & a reducing agent (2-mercaptoethanol). SDS complexes with protein and denatures the protein as well as imparts a negative charge to it. Basic Principle SDS - PAGE is an electrophoresis method that allows protein separation by mass. The medium (also referred to as ′matrix′) is a polyacrylamide-based discontinuous gel . In addition, SDS (sodium dodecyl sulfate ) is used. About 1.4 grams of SDS bind to a gram of protein, corresponding to one SDS molecule per two amino acids.

7. Why SDS PAGE used for proteins? For proteins , Sodium Dodecyl Sulfate ( SDS ) is used to linearize proteins and to negatively charge the proteins . ... As a result, negatively charged proteins will migrate towards the positive electrode and will be fractionated by approximate size during electrophoresis . This procedure is called SDS - PAGE . How does SDS PAGE gel work? SDS - PAGE separates proteins primarily by mass because the ionic detergent SDS denatures and binds to proteins to make them uniformly negatively charged. Thus, when a current is applied, all SDS -bound proteins in a sample will migrate through the gel toward the positively charged electrode.

8. chromatography Chromatography is a physical method of separation that distributes components to separate between two phases, one stationary (stationary phase), the other (the mobile phase) moving in a definite direction. The eluate is the mobile phase leaving the column. ... The eluent is the solvent that carries the analyte. Size exclusion chromatography Size - exclusion chromatography (SEC), also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size , and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers.

9. Basic Principle The number average molecular weight (Mn) is calculated by dividing the total polymer weight by the total number of polymer molecules , using equation (1). The weight average molecular weight (Mw) is calculated using equation (2), which emphasizes the contribution of polymers with larger molecular weights . How does size exclusion chromatography work? Size exclusion chromatography (SEC) separates molecules based on their size by filtration through a gel. ... Small molecules diffuse into the pores and their flow through the column is retarded according to their size , while large molecules do not enter the pores and are eluted in the column's void volume.

10. Gel Filtration chromatography Gel filtration is a technique in which the separation of components is based on the difference in molecular weight or size. It is the simplest and mildest of all the chromatography techniques and separates molecules on the basis of differences in size. Principle . The gel filtration chromatography is based on the molecular size and the hydrodynamic volume of the components. The molecules are separated by the differential exclusion or inclusion of solutes as they pass through the stationary phase containing heteroporous cross-linked polymeric gel or beads. Gel filtration chromatography is an established method for determining the size and molecular mass of proteins. Fractionation is based on the diffusion of molecules into the pores of the resin.

11. How does gel filtration chromatography work? In a gel filtration chromatography column, the stationary phase is composed of a porous matrix, and the mobile phase is the buffer that flows in between the matrix beads. Molecules and complexes that are too large to enter the pores stay in the mobile phase and move through the column with the flow of the buffer.

12. Mass spectroscopy Mass spectrometry (MS) is an analytical technique that measures the mass -to-charge ratio of ions. The results are typically presented as a mass spectrum, a plot of intensity as a function of the mass -to-charge ratio. Basic Principle A mass spectrometer generates multiple ions from the sample under investigation, it then separates them according to their specific mass -to-charge ratio (m/z), and then records the relative abundance of each ion type. What is mass spectroscopy used for? Mass spectrometry is an analytical tool useful for measuring the mass -to-charge ratio (m/z) of one or more molecules present in a sample. These measurements can often be used to calculate the exact molecular weight of the sample components as well.

13. how does it work? A mass spectrometer produces charged particles (ions) from the chemical substances that are to be analyzed. The mass spectrometer then uses electric and magnetic fields to measure the mass ("weight") of the charged particles. tough-cobra-63

14. What are the main characteristics of an enzyme? Speed up chemical reactions. They are required in minute amounts. They are highly specific in their action. They are affected by temperature. They are affected by pH. Some catalyze reversible reactions. Some require coenzymes. They are inhibited by inhibitors.

15. Enzymes speed :- Enzymes are usually helps in increasing the speed of catalysis process. Enzymes speed the rate of a reaction by lowering the amount of activation energy required to reach the transition state, which is always the most difficult step in a reaction. Specificity :- Most of the enzymes are very specific in nature. They act on the their particular related substrate. Specificity is the ability of an enzyme to choose exact substrate from a group of similar chemical molecules. The specificity is actually a molecular recognition mechanism and it operates through the structural and conformational complementarity between enzyme and substrate. Temperature :- Enzyme activity increases as temperature increases, and in turn increases the rate of the reaction. This also means activity decreases at colder temperatures . All enzymes have a range of temperatures when they are active, but there are certain temperatures where they work optimally.

16. pH :- Enzymes are affected by changes in pH . The most favorable pH value - the point where the enzyme is most active - is known as the optimum pH . Extremely high or low pH values generally result in complete loss of activity for most enzymes . Coenzyme :- Coenzymes are small molecules. They cannot by themselves catalyze a reaction but they can help enzymes to do so. In technical terms, coenzymes are organic nonprotein molecules that bind with the protein molecule (apoenzyme) to form the active enzyme (holoenzyme). Unlike the inorganic cofactors, coenzymes are organic molecules. Certain enzymes need coenzymes to bind to the substrate and cause a reaction. Since the coenzymes are changed by the chemical reaction, these are considered to be secondary substrates of the reaction. Inhibitors :- Enzyme inhibitors are substances which bind to the enzyme with resulting loss of activity, without damaging the enzyme's protein structure. These substances which alter the catalytic action of the enzyme and consequently slow down, or in some cases, stop catalysis.

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