Characterization of enkephalin-immunoreactive peptides generated by peptic digestion of rat plasma proteins.
Met-Enkephalin-immunoreactive peptides (MEIP) and Leu-enkephalin-immunoreactive peptides (LEIP), generated from plasma proteins after digestion with pepsin, were characterized with gel filtration, reverse phase HPLC, and isoelectric focusing. Pooled rat plasma was found to generate 170 pmol LEIP and 76 pmol MEIP/ml plasma. Two peaks of enkephalin (enk)-immunoreactive peptides (EIP) were observed after gel filtration of plasma digests; the early eluting peak (which contained 90% of the total LEIP and 75% of the total MEIP) eluted close to, but not in, the void volume of the column, whereas the elution volume of a later eluting peak (which contained 25% of the total MEIP and about 5% of the total LEIP) was close to that of authentic enk. Five peaks of EIP with pI values of 3.7, 5.8, 7.3, 8.0, and 9.4, were observed after isoelectric focusing of plasma digests; with the exception of the pI 3.7 peak, all of these immunoreactive species could be detected with greater efficiency using the Leu-enk RIA. Peptic digestion of rat serum albumin generated a MEIP with the same pI and HPLC retention time as the plasma pI 3.7 MEIP. No other EIP could be generated from rat serum albumin. No immunoreactive peptides were found which coeluted with synthetic Met-enk or its sulfoxide on HPLC; however, a portion of low mol wt LEIP was eluted with the retention time of authentic Leu-enk. With certain exceptions, these results support and extend the original findings of Singer and Carraway concerning the ability of pepsin to generate extremely high concentrations of EIPs from plasma protein(s).
D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004743:Enkephalin, Leucine / Q000097:blood*; D004744:Enkephalin, Methionine / Q000097:blood*; D005260:Female; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008970:Molecular Weight; D010434:Pepsin A / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D012709:Serum Albumin / Q000378:metabolism
D001798:Blood Proteins; D012709:Serum Albumin; D004744:Enkephalin, Methionine; D004743:Enkephalin, Leucine; D010434:Pepsin A