Purification and partial characterization of canine calprotectin.
Calprotectin (CP) is an abundant protein in human neutrophilic granulocytes and macrophages. In humans, serum, urine, and fecal concentrations of neutrophil-derived proteins, such as CP are used as markers of disease activity for conditions associated with increased neutrophil activity, such as inflammatory bowel disease. The aims of the present study were to purify and partially characterize CP in the dog (Canis familiaris) as a prelude to the development of an immunoassay for the quantification of canine serum, urine, and fecal CP in dogs with inflammatory conditions. Leukocytes were isolated from whole blood by dextran sedimentation, and canine CP (cCP) was extracted from the cytosol fraction by repeated freezing--thawing--sonication, followed by further purification using anion- and cation-exchange column chromatography. The overall yield of the purification protocol was 3.7mg cCP per 600ml whole blood. The relative molecular masses of the two proteins representing cCP (cS100A8 and cS100A9) were estimated at 10,340 and 14,628, respectively. Isoelectric focusing revealed two bands with isoelectric points of 6.4 and 6.2 for the heterodimeric protein. The approximate specific absorbance of cCP at 280nm was 0.872 for a 1mg/ml solution. The amino acid sequence of the first 13 N-terminal residues of cS100A8 was Met-Leu-Thr-Glu-Leu-Glu-Ser-Ala-Ile-Asn-Ser-Leu-Ile, whereas the N-terminus of cS100A9 was blocked. Identity of both cS100A8 and cS100A9 was confirmed by tryptic peptide mass fingerprinting followed by peptide sequencing. Antibacterial activity of cCP against Escherichia coli was shown to be concentration-dependent and was reversible upon addition of micromolar amounts of zinc. We conclude that cCP can be successfully purified from canine whole blood using this reproducible, rapid and efficient method.
Heilmann
R M
RM
Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences, 4474 TAMU, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4474, USA.
Suchodolski
J S
JS
Steiner
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D017124:Conserved Sequence; D004285:Dogs; D004926:Escherichia coli / Q000187:drug effects; D006801:Humans; D039841:Leukocyte L1 Antigen Complex / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D013058:Mass Spectrometry; D008970:Molecular Weight; D016415:Sequence Alignment
D000900:Anti-Bacterial Agents; D039841:Leukocyte L1 Antigen Complex