The amino acid 2,6-diaminopimelic acid (DAPA) is a unique component in bacterial cell walls, thus it can be used as an indirect and specific indicator for the measurement of bacterial mass content in such biological materials as feeds or digestive tract contents in ruminants. Further, DAPA’s analogues 2,6-diamino-3-hydroxypimelic acid, together with some diaminopimelic acid, occurs in the cell-wall mucopeptide of certain Actinomycetales. For its discovery, Hoare and Work isolated the chromatographically slow-moving component recognized in hydrolysates of some Actinomycetales from Ampullariella regularis. The results showed that the analyte detected was 2,6-diamino-3-hydroxypimelic acid. This compounds exists four pairs of optical enantiomorphs and these racemic pairs were synthesized by Stewart (1961) and identified as A, B, C and D (shown in Figure 1.) according to the relative configurations of the three asymmetric centers. Further, when comparing the properties of the compound from Ampullariella regularis with the synthesized isomers, it shows that the compound was isomer B.
Figure 1. Configuration of 2,6-diamino-3-hydroxypimelic acid isomers.
These components were converted into their di-DNP derivatives and separated by chromatography. Hence, the relative proportions present in the cell walls of different species were measured. In this way, the bacterial mass content could be detected indirectly.
The traditional method used to quantitate DAPA and 2,6-diamino-3-hydroxypimelic acid is ion-exchange chromatography (IEC) followed by the ninhydrin reaction. This method is reliable and the resolution of the amino acids is reasonable, but the analysis time is long and has a rather limited detectability (c.a. 150 pmol). Furthermore, for this analytical method, an expensive amino acid analyzer is required, and high levels of methionine in analyzed samples would have a big influence on the determination. The alternative method is using charcoal and anionic resin columns which is cheaper but is labor intensive and sensitive to interference from proline.
The use of thin layer chromatography (TLC) for the quantitative analysis of DAPA in biological materials has become widely popular in recent years due to its sensitivity, accuracy and reliability. In Creative Proteomics, whole cell hydrolysates (4N HCl, 100°C, 16 hours) are examined by TLC on cellulose plates for the presence of DAPA isomers or OH-DAP.
Platform
- TLC
Summary
- Detection of isomers of 2,6-diaminopimelic acid and of 2,6-diamino-3-hydroxypimelic acid with TLC
Sample Requirement
- Either a culture of the strain or 5 mg lyophilized cells are needed.
Report
- A detailed technical report will be provided at the end of the whole project, including the experiment procedure, instrument parameters.
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