Molecular characterization of PolyHbCATSOD

A molecular weight distribution analysis is performed using two methods. One is based on gel filtration chromatography on a Sephadex G-200 column equilibrated with 0.1 M Tris-HCl, pH 7.5. Another is

800 630 500 400 310 250 200 150 120 100 80 60 50 40 30 25 20

Molecular Weight (kDa)

Fig. 4.7. Molecular distribution of crosslinked and non-crosslinked ultrapure Hb: Elution profiles of 5-, 10-, 24-h crosslinked PolyHb, PolyHb-SOD-CAT and non-crosslinked bovine SF-Hb are obtained by running 1 ml of the 10x diluted samples on Sephadex G-200 1.6 cm x 70 cm column (VT = 105.5 ml, Vo = 38 ml, equilibrated with 0.1 MTris-HCl, pH 7.5, and eluted at 11.5 ml/h. (Powanda & Chang, 2002.)

800 630 500 400 310 250 200 150 120 100 80 60 50 40 30 25 20

Molecular Weight (kDa)

Fig. 4.7. Molecular distribution of crosslinked and non-crosslinked ultrapure Hb: Elution profiles of 5-, 10-, 24-h crosslinked PolyHb, PolyHb-SOD-CAT and non-crosslinked bovine SF-Hb are obtained by running 1 ml of the 10x diluted samples on Sephadex G-200 1.6 cm x 70 cm column (VT = 105.5 ml, Vo = 38 ml, equilibrated with 0.1 MTris-HCl, pH 7.5, and eluted at 11.5 ml/h. (Powanda & Chang, 2002.)

based on HPLC molecular weight gel filtration analysis on a BioSep Sec S-3000 column (5000-700,000 kD exclusion limits, Phenomenex, Torrance, CA). Since only trace amounts of antioxidant enzymes are used in relation to Hb, the molecular weight distribution of PolyHb-SOD-CAT is the same as that of PolyHb (Fig. 4.7). The ratio of Hb to SOD and CAT (as mg/ml) is typically 1:0.009:0.0045. Only 5% of the enzyme is not crosslinked into the PolyHb-CAT-SOD (Powanda & Chang, 2002) (Table 4.1). There is no CAT or SOD enzyme activity in

Table 4.1 Enzyme Activity after Crosslinked into PolyHB-CAT-SOD

Sample

%SOD Activity Retained

%CAT Activity Retained

Hemoglobin + SOD + Catalase

100

100

PolyHB-SOD-CAT 8:1 Glut:Hb

90

99

PolyHB-SOD-CAT 16:1 Glut:Hb

85

8 or 16:1 Glut:Hb

<1

<1

the PolyHb prepared from ultrapure Hb. Crosslinking CAT, SOD and Hb with a glutaraldehyde: Hb molar ratio of 8:1 retains 90% of SOD activity and 99% of CAT activity. A higher molar ratio of 16:1 leads to a slightly lower enzyme activity as shown in Table 4.1.

Molecular

MW Component

SOD Activity

Catalase Activity

Weight

(%)

(%)

(%)

PolyHb-SOD-CAT sample

>450kDa

77

83 ± 3

82 i 2

100 to 450 kDa

16

7 ± 2

11 i 1

<100kDa

7

4 ± 1

5 i 1

PolyHb sample

>450kDa

77

0

0

100 to 450 kDa

16

0

0

<100kDa

7

0

0

0 0

Post a comment