The chromogenic substrate Bz-Ile-Glu-Gly-Arg-pNA is based on the primary structure preceding the bonds split by factor Xa in bovine prothrombin. Substitution of amino acids in this natural sequence by closely related amino acids has only given inferior substrates. Thus, the natural sequence seems very important. However, we have found that the free γ-carboxyl group of Glu is not indispensable. Substrates with the above structure but having been derivatized on the γ-carboxyl group of Glu, in the form of simple esters or amides, show improved properties. Especially Km of the new substrates compare favourably with our first substrate. Some of the amides also show increased Vmax.These improved properties have made it possible to increase sensitivity, shorten incubation times and lower substrate consumption in several methods where this type of substrate is utilized.

The factor-X_a-sensitive substrate Bz-Ile-Glu-Gly-Arg-p-nitroanilide has been made more sensitive by making ester and amide derivatives of the γ-carboxyl group of the glutamyl residue. The morpholinyl and piperidyl amides react 2.5 times more rapidly with factor X_a.

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In order to improve and facilitate studies of various proteases and their possible interactions with the thromboembolic mechanisms, chromogenic substrates for granulocyte elastase, chymotrypsin, and Cls and Clr have been studied.Substrates of the type Suc-Ala-Ala-Ala-pNA or Ac-Ala-Pro- Ala-pNA (S-2483) are useful for pancreatic elastase but not particularly sensitive towards granulocyte elastase. By placing valine in the PI position, the specificity requirements of granulocyte elastase are better satisfied. Thus pGlu-Pro-Val-pNA (S-2484) was found to be a suitable substrate for this enzyme. In tris buffer pH 8.2 I 0.40 Km was 0.4 mmol/1 with S-2484 and the substrate was approximately 30 times more sensitive than Suc-Ala-Ala-Ala-pNA.The enzyme concentration of 4 nmol/1 gave a change in absorbance per minute of approximately 0.01.In our search for water soluble chromogenic substrates for chymotrypsin, tripeptide derivatives of p-nitroanilin were synthezised where the aliphatic amino acid residues in the P3 position present in most chymotrypsin substrates earlier described were substituted with arginine. In the PI position both tyrosine, phenylalanin and tryptophan were used. The two substrates Sue- and Suc(OMe)-Arg-Pro- Tyr-pNA (S-2558 and S-2586 respectively) were found to be the most potent ones. In tris buffer pH 7.8 I 0.40 and with 3 mmol/1 of Ca2+ Km was 0.05-0.1 mmol/1 and kcat 50-60 per sec. This type of substrates would allow chymotrypsin assays of biological samples without the presence of organic solvents.By studying purified preparations obtained from two different laboratories it was concluded that Cls and Clr have a similar specificity pattern towards pNA-derivatives of tripeptides. In all cases, the substrates split by these preparations have a C-terminal arginine residue. Some already known substrates seem to be sensitive enough to be utilized in biochemical studies of these enzymes.