A high molecular weight form of human urinary urokinase.

A very high molecular weight form of urokinase, of about 100,000 D (VHMr-UK), was isolated from human urine in trace amounts as compared to the well characterized two forms of urokinase, HMr-UK (Mr of about 50,000) and LMr-UK (Mr of about 30,000). This form was demonstrated to be a dimer of HMr-UK, in which no covalent bond is involved, on the basis of the following evidence: by SDS electrophoresis it is dissociated, to the 50,000 D form; by electrophoresis in SDS under reducing conditions it is dissociated, as HMr-UK, to two polypeptide chains of about 30,000 and 20,000 D; by short heating at pH 5 it is quantitatively converted to the 50,000 D form; the kinetic constants towards the alpha-carbobenzoxy-L-lysine-p-nitro-phenylester substrate are the same for HMr-UK and VHMr-UK.

Surface modification of proteins. Activation of monomethoxy-polyethylene glycols by phenylchloroformates and modification of ribonuclease and superoxide dismutase.

A single-step method of activation of monomethoxypolyethylene glycols suitable for its binding to polypeptides and proteins is proposed. Based on the reaction with 2,4,5-trichlorophenylchloroformate or p-nitrophenylchloroformate, it gives reactive PEG-phenylcarbonate derivatives. The PEG intermediate is stable on storage, the activating group is easily quantified,and the reaction with amino acid and proteins proceeds rapidly at pH near neutrality. The PEG derivatization of enzymes with this procedure is less inactivating than those previously reported. Ribonuclease and superoxide dismutase were modified and the effect of (a) bound polymer on clearance time in rats, (b) antibody recognition, and (c) on the enzymatic activity toward low and high molecular weight substrates were studied.

Coupling of monomethoxypolyethyleneglycols to proteins via active esters.

Two alternative methods for the attachment of monomethoxypolyethyleneglycols (PEG) to proteins are proposed; they are based upon the replacement of the hydroxy terminal function of PEG to carboxylate followed by its activation with dicyclohexylcarbodiimide and N-hydroxysuccinimide. The methods, which give more homogeneous product than that employing trichloro-s-triazine as coupling reagent, may also be used for the modification of essential -SH containing enzymes. The attachment of PEG activated via esters was tested with several model proteins and the influence of the extent of modification i. on the biological activity of various enzymes, ii. on the binding capacity for albumin and iii. on the clearance time in rats using superoxide dismutase as model tracer was evaluated. It was also demonstrated that the extent of PEG attachment varies greatly according to the different proteins used.