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APPLICATION NOTE

AN081203-31

StyrosZymeä Papain, Immobilized Enzyme on Simulated Monolith Polymeric Stationary Phase: Effect of Temperature and Linear Velocity on Online Digestion.

The chemical and mechanical stability of STYROSä Simulated Monolith provides the optimum support for the full range of immobilized components such as enzyme.

The present application note shows the practical aspect of the resulting products commercialized by OraChrom under the name StyrosZymeä.

During optimum conditions a StyrosZymeä Papain column can digest over 99 % of a Cytochrome c sample during an online digestion in 4 minutes. The following chromatogram shows the resulting peptides mapped on a STYROSä 2R/XH column placed in tandem after the enzyme column.

Chromatogram 1

Peptide digest from Cytochrome c separated on a STYROSä 2R/XH 4.6 X 250 mm.
Digestion flow rate 0.15 ml/min. Temperature 60 °C.

 

  Operating parameters for the chromatograms.

HPLC System.

Agilent 1100

Columns

StyrosZymeä Papain 4.6 x 33 mm
 STYROSä 2R/XH 4.6 X 250 mm

Mobile Phase For reversed phase.

A: 0.1% TFA in H2O
B: 0.1% TFA in ACN:H2O (95:5)

Digestion buffer.

20 mM Phosphate, 20 mM Cysteine-HCl, 10 mM EDTA, at pH=6.2.

Detection

214 nm

Gradient for Separation

12 to 37 % B in 70 min @ 0.5 ml/min flow rate

Injection volume

8 ml

Sample:

10 mg/ml Cytochrome c from horse heart in 0.1 % TFA.

 

By modifying the temperature of the StyrosZymeä Papain column, or changing the flow rate during the on line digestion, one can alter and control the extent of digestion.

In the first example the temperature was decreased from 60°C to 37°C. As a result the digestion was reduced from 99% to 70% of the total protein injected on the enzyme column.

Chromatogram 2

Peptide digest from Cytochrome c separated on a STYROSä 2R/XH 4.6 X 250 mm.
Digestion flow rate 0.15 ml/min. Temperature 37 °C.

 

By increasing the flow rate from 0.15ml/min to 0.2ml/min during digestion, only 80% of the injected protein is digested.

Chromatogram 3

Peptide digest from Cytochrome c separated on a STYROSä 2R/XH 4.6 X 250 mm.
Digestion flow rate 0.2 ml/min. Temperature 60 °C.
 

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