Purification and analysis of protamine

Protamine, is a cationic peptide derived from fish milt (spermatic cells) and used in medical applications as a carrier for injectable insulin, a heparin antagonist and more recently as an antibacterial ingredient in some food products. To date, several procedures have been proposed for the purification and quantification of protamine. Of course purification of any natural product from a complex matrix necessitates its accurate quantification so that the commercial process may be optimized for recovery, yield and purity. A new pilot scale extraction and purification of protamine from frozen herring milt is described. The fractionation procedure is simple, inexpensive and does not involve the use of time-consuming chromatographic procedures nor organic solvents. Conditions for the optimum extraction and recovery of protamine as both its chloride and sulfate salts is given. Recoveries from frozen herring milt were 1.1% and 1.5% for protamine hydrochloride and protamine sulfate, respectively. Furthermore, the purification procedure was monitored and recoveries optimized using the new affinity chromatographic approach on heparin-agarose. The finished product was freeze-dried and either met or exceeded both the U.S. and E.U. Pharmacopeia standards. Various methods have been reported for the detection and quantification of protamine but few of the methods published to date are highly specific. The present study describes the determination of protamine using a novel fast protein liquid chromatographic separation on an agarose-heparin affinity column. Protamine was quantitatively eluted as a single peak with a buffered salt solution and peaks detected photometrically at 220 nm. Sample preparation was carried out on acid extracts of spiked food samples including (ground beef and coleslaw). The run time including separation and column equilibration was about 42 min and the detection limit for protamine was 30 μg/mL. The affinity technique was found superior in many ways to previously published analytical procedures.