Structural characterization of human hemoglobin crosslinked by bis(3,5-dibromosalicyl) fumarate using mass spectrometric techniques.

Diaspirin crosslinked hemoglobin (DCLHb) was analyzed by mass spectrometric-based techniques to identify the protein modifications effected by the crosslinking reaction with bis(3,5-dibromosalicyl) fumarate. DCLHb consists of two principal components. These components were isolated by size-exclusion chromatography and identified by measurement of their molecular weight using electrospray mass spectrometry and subsequent peptide mass mapping and mass spectrometric sequence analysis of their individual digests. Three major RP-HPLC fractions were observed from the major hemoglobin in DCLHb. Their MWs matched the MW of heme, intact hemoglobin beta-chain, and two hemoglobin alpha-chains crosslinked by a fumarate moiety, respectively. The minor HPLC peaks of DCLHb were also separated, and characterized by mass spectrometric methods. These minor components revealed additional details of the structural nature of covalent modification of DCLHb.

Regulators of Escherichia coli K99 region 1 genes.

Expression of K99 is highly regulated being dependent on 8 K99-specific genes and several host-specific genes including cyclic AMP receptor protein (Crp) and leucine responsive protein (Lrp). The 8 K99-specific genes are organized into 3 separately regulated clusters (regions I-III) with region I and II genes being dependent on Crp. Using TnphoA tagged K99 genes, Lrp was shown to be required for expression of region I genes. Differential methylation of GATC boxes is a common method by which Lrp functions as a regulator. Two GATC boxes are present adjacent to the 5' end of fanA, the first gene. Using the restriction enzymes Dpn I and Mbo I which recognize methylated and non-methylated GATC boxes, respectively, it was shown that differential methylation was not a mechanism regulating K99 expression. Using a gel mobility shift assay and protein extracts from various strains, it was shown that a 625 bp DNA fragment adjacent to the 5' end of fanA bound protein prepared from Lrp+ strains but not from Lrp- strains. While region I genes also require CRP for expression, the same degree of gel shift was observed when the extract was prepared from a Crp- strain. The products of fanA and fanB are believed to be positive regulators. However, protein extracts from strains with or without fanA and fanB caused the same degree of gel shift. Thus, while there are a variety of regulators necessary for region I gene expression, only Lrp or Lrp regulated proteins bind to the promoter region 5' to fanA.