The MICs of the six ovine hemoglobin peptide fractions with mass spectrometry identities were to be confirmed. As a starting point, the RP-HPLC peptide standard curve (see Appendix 2.2) was used to calculate the peptide concentration of each fraction that was required to produce the inhibition zones in Table 6.5. The concentration of peptide in these fractions was determined to be very high, so it was assumed that activity would be retained after a significant dilution, as most active AMPs exert activity in microgram per millilitre concentrations. Each freeze dried fraction was reconstituted in 100ul of 0.01% acetic acid and successive dilutions from this were made for MIC determination by radial diffusion assay, pH 5.5. However, it was found that even at the highest concentration tested, no growth inhibition occurred.
The assay was then repeated using higher peptide concentrations i.e. concentrations in the range of those that initially produced zone clearing. This required fractions from three RP- HPLC runs to be pooled together to give enough material for MIC testing; the exact concentration of these fractions were again determined by the standard curve. From this, dilutions were made for MIC testing and 10ul of each was assayed.
It was determined that the MICs of all six fractions were very high against all three test organisms. Consistent with results from Table 6.5, only fraction 38 exerted activity towards
C.albicans at the highest test concentration, and this was weak activity also. Table 6.7 shows the MIC of each fraction against the three test organisms.
Table 6.7 - MICs of pepsin digested ovine hemoglobin RP-HPLC fractions.
Fraction number MIC range (mg/ml)
E.coli S.aureus C.albicans
20 16-20 25-31 No activitya 38 15-18 12-15 13-16 39 30-37 33-41 No activityb 42 22-28 23-29 No activityc 46 29-36 35-44 No activityd 48 17-21 22-28 No activitye
No activity at highest test concentration of 31mg/mla, 43mg/mlb, 30mg/mlc, 46mg/mld or 33mg/mle.
Single radial diffusion assays were carried out as each assay required pooling three independent RP-HPLC runs to allow for visible zone inhibition. Ideally these assays would be replicated. However, their purpose here was to screen for potential potent AMPs, which can later be synthesized in larger quantities and tested, rather than testing for highly accurate MIC values outright.
Underlay was set at pH 5.5 and fractions were dissolved in 0.01% acetic acid.
Various pH (4.5. 5.5, and 7.4) and reconstitution agents (sodium phosphate, acetic acid, water) were implemented but offered no improvement to MIC values.
6.2.3.5.1 MICs of Synthetic Ovine Hemoglobin Peptides
In this project, a few synthetic ovine hemoglobin peptides were made for antimicrobial testing also, based on literature evidence that suggests strong MICs of these peptides:
x beta-A(140-145) -LAHRYH-
x beta-B(140-145) -LAHKYH-
x beta-B(129-145) -FQKVVAGVANALAHKYH-
x alpha(96-107) -PVNFKLLSHSLL-
In particular, the sequence LAHRYH from bovine hemoglobin, which is conserved in the ovine hemoglobin beta subunit allele A, has been reported as highly active against E.coli, S.enteritidis, L.innocua, and M.luteus, with MIC values ranging from 2 to 10uM (Nedjar- Arroume et al., 2008). Also, -FQKVVAGVANALAHKYH- falls within a widely reported antimicrobial region from the hemoglobin beta subunit. This region is highly conserved between hemoglobin species. These peptides were tested for their MICs against E.coli and
S.aureus, using the same radial diffusion assay at pH5.5 which is used elsewhere in this research. However, only beta-B(129-145) was found to be active at the highest test concentration of 2mg/ml, and this was only against E.coli.
Due to these unexpectedly poor MIC results, another common method for determining antimicrobial activity was performed - the microtitre broth assay. In this method the peptides were serially diluted two-fold in 0.01% acetic acid plus 0.2% BSA, and log-phase cells were added and incubated overnight. The last well with clearing, i.e. no growth, corresponds to the MIC value. However, none of the peptides tested in this research (aside from positive controls) were active under the conditions of this system. This may be due to the presence of BSA, which is a serum protein that may inhibit peptide activity. Nedjar- Arroume et al. (2008) were contacted several times in regards to their MIC values from pepsin digestion of bovine hemoglobin, without response.
It was concluded that none of the ovine hemoglobin digest fractions or synthetic peptides are particularly active against E.coli, S.aureus or C.albicans - only in milligram per millilitre concentrations. To assess why, the structural characteristics of the peptides in each fraction and each synthetic peptide were examined.
Interestingly, towards the end of this research a conference abstract, ‘An Ovine Hemoglobin Fragment Inhibits Porphyromonas gingivalis and Micromonas micros’ by Steward-Tharp et al. (2007) was discovered, and the group was contacted for further information (Brogden, 2011). The group found a 36 amino acid peptide from the C-terminus of the beta chain of sheep hemoglobin (in tracheal lysates) to be active against E.coli. However, when
synthesised, the peptide showed no activity against E.coli, S.aureus, P.aeruginosa,
S.marcescens or A.actinomycetemcomitans. Unusually, the peptide was active in microgram per millilitre concentrations against two oral bacteria, which are typically resistant to
defensins and cathelicidins. The mean MIC values were 250ug/ml and 99ug/ml against
M.micros and P.gingivalis, respectively. No further work was published on this, but perhaps the peptides generated in the research presented in this thesis are also more active towards a different group of microorganisms. Furthermore, it is interesting to note the lack of activity of the synthetic form against E.coli, suggesting that a modification is likely necessary for