The initial rate of haemoglobin digestion peaked at pH 4·0. Above pH 6·0, the rate was no different to controls, which correlated with gel analysis of the 24-h reaction samples; revealing that the 15-kDa haemoglobin doublet was depleted up to pH 6·1 compared to controls (Figure 2). For reactions with albumin a very similar profile was generated, with the fastest initial rate of digestion observed at pH 3·7 (Figure 3). However, the initial rate values obtained were selleckchem approximately fivefold lower than those for the digestion of haemoglobin and consequently much closer to background control values. SDS PAGE analysis confirmed that there was a decrease in the intensity
of intact albumin, accompanied by an increase in lower molecular weight bands presumed to be partially digested albumin, below pH 5·6. It can also be seen that below pH 4·2, albumin digestion occurred in the absence of H-gal-GP, presumably as a result of the acidic conditions Selleckchem PD98059 (Figure 3). Similarly for haemoglobin digestion, the doublet in the 24-h samples corresponding to haemoglobin shows decreased intensity compared to corresponding 0-h samples for enzyme-free controls as well as for reactions containing H-gal-GP at pH conditions below pH 4·2 (Figure 2). Reactions
of H-gal-GP with different concentrations of ovine haemoglobin substrate at pH 5·0 were set up and the increase in free amino groups was monitored by taking samples at regular time intervals. It was assumed that the absorbance value obtained after a 24-h digestion represented a complete turnover of all haemoglobin in the reaction and therefore
was equivalent to the total concentration of haemoglobin in the reaction. The absorbance value of each sample from all time points was used to estimate its concentration of haemoglobin. These concentration estimates were then plotted against time to obtain a turnover rate per second (v). This rate was plotted against the total concentration of haemoglobin in the reaction to produce the Michaelis–Menton curve which gave a kcat of 0·03 s−1 and a KM of 29 μm (Figure 4). The rate of digestion of ovine haemoglobin was monitored Cetuximab in vitro as before except that the H-gal-GP was pre-incubated with serum IgG obtained from sheep which had been successfully vaccinated with H-gal-GP (pIgG – see Table 1) or from control sheep immunized with adjuvant alone (cIgG – see Table 1). Different pH conditions under which IgG bound to H-gal-GP (with pre-incubation at pH 7·4 followed by reaction at pH 4·0 and with pre-incubation and reaction both at pH 4·0 as described in the Materials and Methods) were tested before the detection of IgG inhibition at pH 5·0 (data not shown). For inhibition experiments carried out with both the IgG pre-incubation and subsequent reactions held at pH 5·0, H-gal-GP was incubated with either pA, pIgG, cIgG or buffer.