Protamine sulphate

Protamine sulphate (from herring sperm) [9007-31-2] (a]p -85.5 (satd H2O). A strongly basic protein (white powder) with pKa values of 7.4-8.0 used to ppte nucleic acids from crude protein extracts. It dissolved to the extent of 1.25% in H2O. It is freely soluble in hot H2O but separates as an oil on cooling. It has been purified by chromatography on an IRA-400 ion-exchange resin in the SO form and washed with dilute H2SO4. Eluates are freeze-dried under high vacuum below 20°. This method is used to convert proteamine and protamine hydrochloride to the sulphate. [UV Rasmussen Z physiol Chem 224 97 1934, Ando and Sawada J Biochem Tokyo 49 252 1961, Felix and Hashimoto Z physiol Chem 330 205 1963]... 

A-Acetyl neuraminic acid aldolase [from Clostridium perfringens, A-acetylneuraminic acid pyruvate lyase] [9027-60-5] [EC 4.1.3.3]. Purified by extraction with H20, protamine pptn, (NH4)2S04 pptn, Me2CO pptn, acid treatment at pH 5.7 and pptn at pH 4.5. The equilibrium constant for pyruvate + n-acetyl-D-mannosamine ++ /V-acetylneuraminidate at 37° is 0.64. The Km for A-acetylneuraminic acid is 3.9mM in phosphate at pH 7.2 and 37°. [Comb and Roseman Methods in Enzymology 5 391 1962). The enzyme from Hogg kidney (cortex) has been purified 1700 fold by extraction with H20, protamine sulphate pptn, (NH4)2S04 pptn, heat treatment between 60-80°, a second (NH4)2S04 pptn and starch gel electrophoresis. The Km for A-acetylneuraminic acid is 1.5mM. [Brunetti et al. JBC 237 2447 1962). 

The activation of BSSL is specific to primary bile salts. Bile salts, secondary as well as primary, protect BSSL against inactivation by intestinal proteinases. BSSL is inactivated by heating at 50°C for 1 h, but sodium taurocholate prevents loss of activity. The enzyme is stable in buffer for 1 h at 37°C between pH 3.5 and 9. It is inhibited by blood serum, 1M NaCl, protamine sulphate, eserine and diisopropylfluorophosphate (Hernell, 1975 Blackberg and Hernell, 1983). 

Heparin antagonism. Heparin effects wear off so rapidly that an antagonist is seldom required except after extracorporeal perfusion for heart surgery. Protamine, a protein obtained from fish sperm, reverses the anticoagulant action of heparin, when antagonism is needed. It is as strongly basic as heparin is acidic, which explains its immediate action. Protamine sulphate, 1 mg by slow i.v. injection, neutralises about 100 units of heparin derived from mucosa (mucous) or 80 units of heparin from lung ... 

Doolan L, McKenzie I, Krafchek J, Parsons B, Bnxton B. Protamine sulphate hyperserrsitivity. Anaesth Intensive Care 1981 9(2) 147-9. 

Suspension for Mixture of fast Mixtard (Novo Protamine sulphate, zinc. 

The drug is a sterile suspension of Zinc-insulin crystals and protamine sulphate in buffered water for injection, usually combined in such a fashion that the solidphase of the suspension essentially comprises of crystals composed of insulin, protamine and zinc. 

The protamine sulphate is usually prepared from the sperm or from the mature testes of fish belonging to the genera Oncorhynchus Suckley, or Salmo Linne (Family Salmonidae). 

The drug is a sterile suspension of insulin in buffered water for injection, that has been adequately modified by the addition of zinc chloride (ZnCl2) and protamine sulphate. The protamine snlphate... 

Dunne M, Bibby DC, Jones JC, Cndmore S. Encapsulation of protamine sulphate compacted DNA in polylactide and polylactide-co-glycolide microparticles. Journal of Controlled Release. September 19, 2003 92(l-2) 209-219. PnbMed PMID 14499198. 

Chromatbim Homogenizer Protamine sulphate 1800> Disc electrophoresis Evans et al. (1973)... 

The B.P.C. includes a method for the determination of protamine sulphate on its capacity to neutralise heparin in which the end-point is determined biochemically. 

Make a solution of the protamine sulphate in saline containing 1 mg per ml. To each of ten tubes add 2 5 ml of plasma, maintain at 37° and to the first nine tubes add 0 5 ml of diluted sample. To the tenth tube add 2 0 ml of saline and 0 5 ml of calcium chloride-thrombokinase solution. Determine the time for fibrin threads to appear on a wire loop drawn through the mixture. 

To the remaining tubes add respectively 0 43, 0 45, 0 47, 0 49, 0 50, 0 51, 0-53, 0-55 and 0-57 ml of heparin solution diluted in each case to 1 5 ml with saline. Add to each tube 0 5 ml of calcium chloride-thrombokinase solution and record the coagulation time. The percentage of protamine sulphate is given by the formula a/Q S X 100 where a is the maximum volume of heparin solution which does not prolong clotting time. 

The B.P.C. determination is based on the assumption that 1 mg protamine sulphate neutralises 86 units of heparin. There is little foundation for this assumption and in fact the amount of heparin neutralised depends on the sample of heparin used. For this reason simultaneous comparison should be made using a standard preparation of protamine sulphate. 

In a test of this kind it is not essential to assess the presence of excess heparin by means of its biological activity any other property of heparin, e.g. its ability to combine with toluidine blue, would serve equally well. Birkinshaw and Smith showed that when the protamine sulphate is in excess the spots made on paper (Whatman No. 1) and stained with bromo-... 

Make an aqueous solution of protamine sulphate standard containing 1 mg per ml and similar solutions of the sample under test. Into two series of tubes pipette 1 ml of these solutions and to successive tubes add quantities of heparin in amounts differing by 2 units and contained in 1 ml water. Spot approximately 0 01 ml from each tube on Whatman No. 1 paper pencilling the outline of the wetted area. Allow to dry and stain for five minutes in a 0 02 per cent solution of bromocresol green. Wash three times for five minutes in 2 per cent w/v acetic acid solution. Allow to dry and observe the activity of the sample in terms of the standard. This is proportional to the respective maximal concentrations of heparin which still permit the production of a compact stained spot. 

Hayano has used a phosphate buffer containing KCl and EDTA to extract phosphodiesterases from a forest soil. After dialysis and concentration of the extracts, and precipitation of the enzymes and coextracted coloured humic compounds by ammonium sulphate, solutions of the phosphodiesterases were further purified by addition of protamine sulphate and a second dialysis treatment. Removal of flocculated humic material was accomplished with only small losses of phosphodiesterase activity. Per gram of soil, extracted enzymic activity was curvilinearly related to extractant volume, indicative perhaps of desorption of enzyme ionically bound to soil surfaces. The partially-purified enzyme retained activity after heating solutions for lOmin at 50 C, but was completely inactivated at 80°C. 

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