Process for Sulphation and Phosphorylation of Proteins and Peptides

The following examples are provided to illustrate the method of the present invention but they are in no way meant to limit its scope.

20 mg of porcine sodium insulin was dissolved in a mixture of 905 .mu.l of dry dimethylformamide and 50 .mu.l of concentrated sulfuric acid at 4.degree. C. 80 mg. of N,N' dicyclohexyl carbodiimide (DCC) dissolved in 100 .mu.l of DMF was added to this mixture and the solution was shaken overnight at 4.degree. C. Cracked ice (approx. 1 g was added and the pH was adjusted to 7.5 with 1ON NaOH. The solution was brought to 5 ml and centrifuged. The supernate was extensively dialysed against 0.05M ammonium bicarbonate, pH=7.5, and lyophilized.

Hypoglycemic potency of the modified insulin was measured as described in "The United States Pharmacopeia", Mark Publishing Co., Vol. 18, pp. 883-884 (1964). Bioactivity was 62% as compared to unmodified insulin.

The tendency of modified insulin to aggregate was assessed by measuring the turbidity of the solution. This method is based on the work of Leach and Scheraga (S. J. Leach and H. A. Scheraga (1964) J. Amer. Chem. Soc. 82, 4790-4792) who stated that absorbancy observed in insulin solutions between 350 and 600 nm is due to light scattering of aggregated particles. Briefly, 10 mg of sample was dissolved in 2 ml of isotonic phosphate buffered saline (PBS) and the solution was passed 50 times through a 3 cc plastic syringe (B-D Plastipak, Luer Lok Type) equipped with a 26G 3/8" hypodermic needle. After deaeration under vacuo for 60 min. 1 ml of this solution was diluted with 1 ml of PBS, and the turbidity was measured on a Hewlett Packard spectrophotometer type HP 8590, utilizing as a blank a non-aggregated sample diluted by 1 volume of 8M guanidine hydrochloride. Aggregation was measured by the difference in absorbance determined at 400 nm for the two samples. Non-aggregated samples gave absorbance values of 0 to 0.1 Absorbance Unit(A), Values above 0.3 were considered to be aggregated. Utilizing this assay the turbidity of this modified insulin dissolved in PBS was 0.08 A. In contrast unmodified porcine sodium insulin dissolved in distilled water has an absorbance of 0.51 A when treated according to the above procedure.

Example 1 was repeated with different amounts of N,N'-dicyclohexyl carbodiimide dissolved in 100 .mu.l of dry dimethyl formamide added to 20 mg of porcine sodium insulin dissolved in the mixture of 950 .mu.l DMF and 50 .mu.l of concentrated sulphuric acid at 4.degree. C.

The modified insulin samples were subjected to gel electrophoresis and densitometric evaluation. Briefly, gel electrophoresis was carried out by dissolving 2 mg of samples (sulphated or unmodified insulin) in 7M urea. 20 .mu.l of this solution was analyzed by electrophoresis at pH-8.3 on polyacrylamide gels containing 25% w/v acrylamide and 0.15% w/v bis-acrylamide under the experimental conditions described by Davis (Baruch J. Davis (1964) Annals of the New York Academy of Sciences, Vol. 121, pp. 404-427). The gels were stained with Coomassie Brilliant Blue R and destained with 10% w/v trichloracetic acid. The R.sub.f values were calculated from the migration distances and were divided by the R.sub.f of unmodified insulin.

Densitometric evaluation was carried out by cutting the individual lanes of the gels and determining the scan with a Beckman ACTA III Spectrophotometer equipped with a type 198402 Gel Scanner Accessory, in silica cuvettes at 590 nm. The peaks were integrated manually and the relative quantities of the products were expressed as % of the total sum of the integral values.

The results of this experiment are summarized in Table 1.

100 mg of porcine sodium insulin was dissolved in the mixture of 4.75 ml of DMF and 0.25 ml of concentrated sulfuric acid at 4.degree. C. The solution was divided into portions of 1 ml. The following reagents were subsequently added to individual tubes:

The samples were proceed as described in Example 1 (except that centrifugation was omitted), then subjected to gel-electrophoresis and densitometric quantitation. Results are summarized in Table 2.

20 mg of porcine sodium insulin was dissolved in the mixture of 950 .mu.l of dry DMF and 50 .mu.l of concentrated phosphoric acid prepared by heating 85% aqueous phosphoric acid to 150.degree.-160.degree. C. for about 4-5 hours. DCC (60 mg) dissolved in 100 .mu.g of DMF was added to this solution, and the reaction mixture was shaken at 4.degree. C. overnight. The sample isolated as described in Example 1 had a bioactivity of 85% as compared to unmodified insulin. Electrophoresis of the sample showed multiple bands bearing more negative charge than unmodified insulin.

20 mg of porcine sodium insulin was dissolved in the mixture of 950 .mu.l of dry DMF and 50 .mu.l of phosphoric acid at 4.degree. C. as described above. POCL.sub.3 (5 .mu.l diluted with 15 .mu.l of DMF) was added and the reaction was carried out as described in Example 4. Electrophoresis of the sample showed 5 bands more negatively charged than unmodified insulin.