4.3.7.1 Sodium dodecyl sulphate polyacrylamide electrophoresis (SDS-PAGE)
The void volume fractions of the Sephadex G-100 column (Fig. 4.5), presumably containing the polysialylated catalase, were subjected to SDS-PAGE together with controls of native enzyme and zero time reaction mixture and results are shown in Fig. 4.9. As anticipated (Aebi, 1983), both controls exibit sharp bands of dissociated protomers of about 60 kDa under denaturing conditions. In contrast, the enzyme patterns in the void volume fractions appeared broad in shape suggesting (Carlsson, 1993) microheterogeneity as a result of the carbohydrate chains, thus supporting the presence of a neoglycoprotein.
It is well established (Segrest & Jackson, 1972) that because of the weak affinity of SDS for the carbohydrate chains of glycoproteins, estimation of their molecular weight by SDS-PAGE is not accurate. However, the negatively charged sialic acid residues are expected (Segrest & Jackson, 1972; Leach et al., 1980) to
partially compensate for the reduced SDS binding, thus rendering the measurement of molecular weight more precise. This was estimated to be in the region of 54.6 ± 1.1 to 96.1 ± 3 . 0 kDa for the polysialylated catalase monomers. Observed (Fig. 4.9) higher molecular weight bands («140 kDa) on the other hand, are likely to represent cross- linked catalase monomers formed through the function of CA molecules as dialdehydes. In this respect, a-2->8 linkages are, under the mild conditions used here, resistant to periodate oxidation (Lifely et al., 1986) and the activated CA would therefore be expected to have essentially one terminal aldehyde group at C? of the non reducing end. However, the other ketonic group at the reducing end, although less reactive because of its involvement in the hemiketal linkage, is in a tautomeric equilibrium and could potentially react with the amino groups of the enzyme. This possibility is supported by the formation of a conjugate (at much lower yields than observed with activated CA) when non-oxidized CA is used in the reaction (Table 4.3).
Gel electrophoresis of native asparaginase (ammonium sulphate precipitates after extensive dialysis) showed a single band, with an estimated molecular weight of 34 kDa (Fig. 4.10, lane a) that is consistent with that (37.5 kDa) reported by Cammack et al. (1972) for the enzyme’s subunit. Even after incubation for 48 h under reaction conditions but in the absence of reagents, asparaginase retained the same single band in SDS-PAGE and no lower MW degradation products were evident (lane b). As with catalase, all the reaction mixtures at time zero (lanes c, f and h) showed a distinct band of dissociated monomers, while after polysialylation the microheterogeneity of the enzyme, on account of the grafting of CA, was demonstrated by the yield of diffuse bands (lanes d, g and i). Differences in the intensities of the protein bands were due to uneven loading of the gel and thus the MW range for each degree of asparaginase modification could not be accurately estimated.
- 2 1 2 - 170 - 116 - 7 6 - 5 3 a b c d e
Fig. 4.9 SDS-PAGE o f native and polysialylated catalase. Fractions eluted in the void volume o f a Sephadex G-100 column (Fig. 4.5B) were compared to the native enzyme and reaction mixture at zero time. Lanes correspond to eluted fractions 12 and 13 (a, b); Pharmacia high molecular weight markers (c); eluted fractions 14 and 15 (d, e); native catalase (f); zero time reaction mixture (g). Numbers to the right indicate the molecular mass (kDa) o f the markers. Gel (pre-cast, homogeneous 12.5% polyacrylamide) was run in a Pharmacia Phast System and stained with Coomassie blue. For other details see Chapter 2.
- 116 - 9 7 -66 m m # - 2 0 5 - 2 9
Fig. 4.10 SDS-PAGE o f native and polysialylated asparaginase. Gel was loaded with the following; (a), native asparaginase; (b), native asparaginase subjected to reaction conditions for 48 h; (c), preparation A, zero time; (d), preparation A, 48 h reaction; (e) Sigma broad range m olecular weight markers; (f), preparation B, zero time; (g), preparation B, 48 h reaction; (h), preparation C, zero time; (i), preparation C, 48 h reaction; (j), Sigma broad range molecular weight markers. Numbers to the right indicate the m olecular mass (kDa) o f the markers. Gel (12.5% polyacrylamide) was run in a vertical electrophoresis unit (Anachem, UK) and silver stained. For other details see Chapter 2.
A similar abnormal migration on SDS-PAGE (continuous smears or multiple bands) has also been observed after derivatization of proteins with PEG. This was attributed to the highly heterogenous nature of the pegylated proteins (Kunitani et al., 1991), not only in terms of MW (dependent on the degree of modification and on the polydispersity of PEG) but also charge (different numbers of lysine residues derivatized result in charge heterogeneity) and shape (attachment of PEG chains at distinct locations of the protein leads to erratic SDS binding). Reduced electrophoretic mobility of pegylated albumin was on the other hand, attributed to the entanglement of PEG strands with polyacrylamide. However, glycosylated proteins that usually also migrate at a slower rate in SDS-PAGE (Segrest & Jackson, 1972) (resulting in erroneously high MW estimations) do so purely because of the reduced binding of the detergent to the carbohydrate chains (Leach et al., 1980).
4.3.7.2 Isoelectric focusing (lEF)
The polysialylation of the lysine surface residues of the enzymes would presumably be accompanied by a decrease in the isoelectric point (pi) of the neoglycoprotein. The in vivo half-life of enzymes has been related by several authors (Holcenberg et al., 1975; Mashbum & Landin, 1970; Rutter & Wade, 1971) to their pi and generally , the lower the pi the more the enzyme is said to persist in circulation. Rutter & Wade (1971) for instance, chemically modified E. carotovora asparaginase to obtain a series of different isoelectric points, ranging fi"om 3.3 to 9.7. Enzymes were then injected intravenously in rabbits and blood clearance was determined, pi values between 5.0 and 6.0 found to lead to the highest half-lives but, below these pi values, the rate of removal of the enzymes from the circulation increased again. The authors
1 0 “I
Q .