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    Methods of Enzyme Purification

    Uploaded by

    Dawlat Salama
    This document outlines several common methods for purifying enzymes, including salt precipitation, ion exchange chromatography, gel permeation chromatography, hydrophobic interaction chromatography, affinity chromatography, and immunoaffinity chromatography. It also describes methods for assessing purity, such as SDS polyacrylamide gel electrophoresis, and defines units of enzyme activity like specific activity, enrichment, and percent purity and recovery.

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    Methods of Enzyme Purification

    Uploaded by

    Dawlat Salama
    296 views2 pages
    This document outlines several common methods for purifying enzymes, including salt precipitation, ion exchange chromatography, gel permeation chromatography, hydrophobic interaction chromatography, affinity chromatography, and immunoaffinity chromatography. It also describes methods for assessing purity, such as SDS polyacrylamide gel electrophoresis, and defines units of enzyme activity like specific activity, enrichment, and percent purity and recovery.

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    Enzyme

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    This document outlines several common methods for purifying enzymes, including salt precipitation, ion exchange chromatography, gel permeation chromatography, hydrophobic interaction chromatography, affinity chromatography, and immunoaffinity chromatography. It also describes methods for assessing purity, such as SDS polyacrylamide gel electrophoresis, and defines units of enzyme activity like specific activity, enrichment, and percent purity and recovery.

    Copyright:

    © All Rights Reserved
    Download as DOC, PDF, TXT or read online from Scribd
    Download as doc, pdf, or txt
    296 views2 pages

    Methods of Enzyme Purification

    Uploaded by

    Dawlat Salama
    This document outlines several common methods for purifying enzymes, including salt precipitation, ion exchange chromatography, gel permeation chromatography, hydrophobic interaction chromatography, affinity chromatography, and immunoaffinity chromatography. It also describes methods for assessing purity, such as SDS polyacrylamide gel electrophoresis, and defines units of enzyme activity like specific activity, enrichment, and percent purity and recovery.

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    © All Rights Reserved
    Download as DOC, PDF, TXT or read online from Scribd
    Download as doc, pdf, or txt
    of 2

    Methods of enzyme purification

    1. salt precipitation

    2. Chromatographic methods
    i. ion exchange (cationic or anionic )
    separation based on charge; the greater the net charge, the more strong
    the interaction ; elute by increasing ionic strength of buffer or altering
    pH

    ii. gel permeation (size exclusion) chromatography:


    separation based on size; larger proteins elute first

    iii. Hydrophobic interaction chromatography:


    separation based on hydrophobicity of protein surface; elute by
    decreasing polarity of buffer ; more hydrophobic bind more strongly and
    elute last.

    iv. affinity chromatography:


    separation based on specific biological interaction; eg enzyme –
    substrate recognition; elute with substrate

    v. immunoaffinity chromatography:
    separation based on specificity of antibody recognition of peptide
    sequence; elute by lowering pH to 2-3 to disrupt antibody – peptide
    interaction. Neutralize quickly to avoid loss of activity.

    Assessment of purity

     SDS polyacrylamide gel electrophoresis

     enrichment

     N-group analysis

    Units of enzyme activity


     Activity: amount of product produced per unit time (eg. ?mols /
    sec)

     Specific activity: amount of product produced per unit time per mg


    protein

    (eg. mmols / sec / mg)


     Enrichment: SA of fraction / SA of starting material

     Maximum possible enrichment: 100 / % of total protein


    represented by protein of interest

     % purity : actual enrichment / maximum possible enrichment

     % recovery (yield) : total activity of fraction / total starting


    activity

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