Heparin-agarose

Figure 2. Elution profile (A) of the EcoYA mtase from a heparin agarose chromatography column showing the presence of two peaks, the major peak being the M2S, form and the minor peak being M,S,. Elution profile (B) formed by reapplying the smaller of the two peaks from elution run A to the heparin agarose column showing the re-equilibration of the mtase into M2S, and MjS forms. % transmission at 280nm was monitored (Dryden et al, 1993).

Dialyze separate BIGl and BIG2 pools overnight against 2 L of buffer A before chromatography on heparin-agarose (1-cm diameter column. 

Heparin Agarose 6XL 6% cross-linked agarose Heparin n a. 

The temperature at which affinity chromatography is performed has been shown to be critical for the isolation of ribosomes on heparin-agarose derivatives the ribosomes are retained at 4 °C but do not bind at 22... 

The purification of liprotein lipase (LPL) from rat adipose tissue [84] and hen adipose tissue [85], respectively, has been described using an affinity column prepared by insolubilization of heparin on agarose. In the case of hen adipose LPL elution was achieved with 1.16 M NaCl and 50% recovery achieved with 80-fold purification. 

Dermatan sulfate is known to specifically catalyze thrombin inhibition by the plasmatic inhibitor heparin cofactor II (HCII). Dermatan sulfate has been immobilized on a dextran- or agarose-coated silica matrix. These systems were tested as high-performance chromatographic supports for the purification of HCII from human plasma. The eluted HCII was obtained with no contamination of ATIII, the other main thrombin inhibitor [16]. 

Figure 2. Electrophoretic migration patterns of commercial heparins. The cathode and the point of application are considerably to the left of each pattern, the anode is to the right. Separation in agarose on the basis of charge density, in polyacrylamide on the basis of molecular size, and in LKB ampholyte on the basis of pH and chain length, producing a specific insoluble complex with ampholyte. (2)...

The degradation of heparin by the reactor is a multistep process. Heparin and the heparin-antithrombin complex must first diffuse from the bulk phase to the surface of the immobilized enzyme particle. The two species diffuse into the agarose particles where they encounter immobilized heparinase. The heparin-anti thrombin complex is assumed to be sterically inhibited from binding to immobilized heparinase, and under these conditions only unbound heparin is enzymatically degraded. As unbound heparin is consumed, heparin dissociates from the heparin-antithrombin complex to generate more free heparin. The breakdown of heparin is given by the following chemical reaction ... 

Figure 4. Immobilization of N-acetulated diaminoalkane agarose gels using N-ethyl-5-phenylisoxazolium-3f -sulfonate (Woodward s Reagent K) activated heparin carboxylic groups.

Table III. Heparin Immobilization onto Agarose Gels...

Figure 9. APTT vs. spacer unit carbon number results for heparin immobilized via carboxylic groups to diaminoalkane agarose gels. Key —, the baseline APTT (i.e.y unheparinized plasma) , respective control substrate APTT (i.e., untreated diaminoalkane agarose gels) and O, respective heparin immobilized gels.

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