Chemical synthesis, purification, and characterization of two inflammatory proteins, neutrophil activating peptide 1 (interleukin-8) and neutrophil activating peptide.
Two recently identified pro-inflammatory proteins, namely, neutrophil activating peptide 1 (NAP-1) [also termed interleukin-8 (IL-8)] and NAP-2, were chemically synthesized, purified, and characterized. The fully protected NAP-1/IL-8 (72 residues) and NAP-2 (70 residues) peptide chains were assembled by automated solid-phase methods with average stepwise yields of 99.5 and 99.3%, resulting in overall chain assembly yields of 70 and 62%, respectively. Deprotection resulted in crude products, which were allowed to fold by air oxidation, and were purified by two cycles of reverse-phase high-pressure liquid chromatography, yielding 27 mg of NAP-1/IL-8 and 22 mg of NAP-2. Purity was established by reverse-phase high-pressure liquid chromatography and isoelectric focusing, and the primary structures of the purified products were verified by using mass spectrometry and Edman sequencing methods. Synthetic and recombinant NAP-1/IL-8 were equally active on human neutrophil granulocytes as determined by measuring the induction of cytosolic free calcium, elastase release, and chemotaxis. Synthetic NAP-2 was equivalent to purified natural NAP-2 in the elastase release and calcium mobilization assays, but it was consistently less potent (3-5-fold) as a stimulus of chemotaxis, perhaps indicative of additional chemotactic components in the natural preparation. The results indicate that by chemical synthesis these cytokines can be obtained in purity and quantities suitable for further structural analysis, as well as functional studies both in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
Clark-Lewis
I
I
Biomedical Research Centre, University of British Columbia, Vancouver.
Moser
B
B
Walz
A
A
Baggiolini
M
M
Scott
G J
GJ
Aebersold
R
R
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002634:Chemotaxis, Leukocyte; D002851:Chromatography, High Pressure Liquid; D016209:Interleukin-8 / Q000138:chemical synthesis* / Q000235:genetics / Q000302:isolation & purification; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000235:genetics / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis* / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation
D016209:Interleukin-8; D010446:Peptide Fragments; D010455:Peptides; C009862:connective tissue-activating peptide; D010196:Pancreatic Elastase; D002118:Calcium