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Abstract
Radioiodinated oLH α and oLH ß subunits were fractionated with the aid of high performance liquid chromatography (HPLC) using a Waters Protein Pak DEAE 5PW anion exchange column. The content of these subfractions differed in their binding maxima to their respective subunit antisera. An increase of the pH from 6.5 to 7.5 and 8.5 affected the chromatographic profile of 8-week-old radioiodinated α-subunit. Overall, material from the various radioactive peaks exhibited binding to ß-subunit antiserum in the range of 32.0% - 81.0%, depending on the storage time of the tracer and the pH. Shifting strategies, we either applied the labelled subunits to a Pharmacia gel filtration column or subjected them to cellulose adsorption prior to HPLC. The radioiodinated α- and β-subunits subjected to HPLC after gel filtration were both eluted in only one peak with respective immunoreactivities of 46.6% and 73.2%.
When radioiodinated β-subunit was applied first to a cellulose column and then to HPLC, the chromatographic profile showed two radioactive peaks with retention times of 5 min (73.2% immunoreactivity) and 7.5 min (43.0% immunoreactivity), respectively.
It was concluded that an 8-week-old-tracer i s useful in such studies, owing to its highly stable immunoreactivity after repurification on an anion exchange HPLC.