StyrosZyme™ Papain: digestion at pH 6.2
The protein is digested during an 8 min digestion in a 4.6 x 33 mm immobilized Papain column. The digests are then deposited on a STYROS™ 2R/XH (4.6 X 250 mm) polymeric column and subsequently mapped.

Contents:

• Product Description
• Overview of On-line Digestion
• General Considerations

• Product Description: 

StyrosZymeä Papain is a monolithic, fully uniform hydrophilic polymer based packing with Papain covalently tethered to the surface of a Simulated Monolith Bed.
It is intended to perform online digestion of protein in a flow through setting.

The base matrix that is fully pervious, is made of highly crosslinked poly(styrene-divinylbenzene).

Our proprietary polymerization technique ensures that the beads are not brittle and as a result, free of any leachables. The unique macroporous structure takes full advantage of the inner bead’s surface area, making it possible to run high speed, high resolution separations without any mass transfer restrictions.

The polystyrenic matrix is rendered hydrophilic through a covalently bound coating.

The packed cartridges offer extremely high-pressure tolerance, and can be used routinely up to 2,000 psi.

The advantages offered by immobilized enzyme in a flow through setting are numerous:

·         Digestion time is reduced to a few minutes as compared to hours.

·         The enzyme cartridge can be used as a direct inlet to either a LC or a MS system for the analysis of the resulting peptides, substantially reducing and simplifying the sample handling process and allowing it to be fully automated.

·         The extent of digestion can be controlled by changing the flow rate and the temperature. It can also be made fully reproducible.

·         The immobilized enzyme displays high stability towards its working range pH’s, organic solvents, high flow rates, temperatures and back pressures.

·         The possibility of using fast flow rates allows the cartridge to be reconditioned quickly, further reducing the process time.

·         Due to the absence of contact in the immobilized format between enzyme molecules, no auto digestion occurs.

StyrosZymeä Papain cartridges are offered in a number of formats to provide a wider selection

Technical Specifications

• Overview of On-line Digestion:

StyrosZymeä columns can be used in series with a reversed phase column followed by a mass spectrometer or as a direct inlet to a MS.

Keep in mind that StyrosZymeä columns can tolerate backpressures of up to 2,000 psi. It is therefore possible to equilibrate and clean them in a very short period of time by running high flow rates.

 The digestion-mapping process involves the following steps:

·        Equilibration: Each column is equilibrated with the appropriate buffer solutions.

·       Loading: The protein of interest is injected onto the StyrosZymeä enzyme reactor column.

·      Digestion: Using the optimum residency time and temperature, the protein is fully digested on-line. The resulting peptides are dumped on the reversed phase column.

·        Separation: The hydrophobically bound peptides are then eluted from the reversed phase column with the option of being further introduced to an MS.

·        Cleanup and re-equilibration: Each column is then eluted with the appropriate buffer.

Papain is very useful in the preparation of immunoglobulin fragments such as Fab and Fc fragments from IgG class antibodies.

The reversed phase column set in tandem for desalting or mapping of the digested fragments must tolerate non-neutral pH’s. STYROSä 2R/XH are highly recommended especially when the effluents are used to feed a mass spectrometer.

The presence of some denaturing agents such TFA or 3M guanidine.HCl promotes the digestion. The proteins that are difficult to digest in solution will be difficult to digest on line. It is necessary to assist the unfolding of the protein by denaturing agents and prevent the refolding by using reducing or alkylating agents.

• General Considerations:

Enzyme digestion varies with proteins as well as the operating conditions such as temperature, flow rate and pH.

By controlling these variables, it is possible to control the extent of digestion. The size of the peptide fragments changes with the temperature as well as the flow rate.

Papain is a single polypeptide with an isoelectric point of 9.6 and an optimum pH range of 6-7 for enzymatic activity.

As a sulfhydryl protease, Papain is not reactive in its native form and needs to be activated by a mild disulfide reducing agent such as Cysteine.

The enzyme catalyzes the hydrolysis of a multitude of peptide, amide and ester linkages. At the hydrolysis point, the carboxyl side of amino acid residue should be Arginine, Glutamine, Glycine, Histidine, Lysine or Tyrosine. The amino side amino acid next to this residue should have a non-polar side chain.

Papain is very useful in the preparation of immunoglobulin fragments such as Fab and Fc fragments from IgG class antibodies.

The reversed phase column set in tandem for desalting or mapping of the digested fragments must tolerate non-neutral pH’s. STYROSä 2R/XH are highly recommended especially when the effluents are used to feed a mass spectrometer.

The presence of some denaturing agents such TFA or 3M guanidine.HCl promotes the digestion. The proteins that are difficult to digest in solution will be difficult to digest on line. It is necessary to assist the unfolding of the protein by denaturing agents and prevent the refolding by using reducing or alkylating agents.

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