A comparison between SDS-PAGE and size exclusion chromatography as analytical methods for determining product composition in protein conjugation reactions

Horseradish peroxidase (HRP) was conjugated with bovine serum albumin (BSA) or human α₁-proteinase inhibitor (α₁-PI). The enzyme was maleimidylated using N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and then allowed to react with thiolated BSA or reduced α₁-PI. The conjugation products were analysed both by SDS-PAGE and size exclusion chromatography (SEC) on Sephadex G200. The two methods of evaluating conjugative processes were compared with respect to information provided in relation to the behaviour of the products in solution. The results showed that neither SDS-PAGE nor SEC alone provides sufficient information about conjugate structure. The basic conjugate units observed in electrophoresis tend to form dimeric or higher-order aggregates under gel chromatographic conditions.