Inter-a-trypsin inhibitor (ITI) is a complex protein made up of a light chain so-called bikunin and two heavy chains (apparent Mr values 96000 and 86000 in SDS/PAGE in non-reducing conditions). By sequence analysis, we clearly identified those two components as HI and H 2 , respectively.We demonstrate that alkaline treatment (50mM NaOH during 5 min at room temperature) as well as chondroitinase digestion both lead to the dissociation of ITI. The conditions used for alkaline treatment were previously reported for cleavage of the covalent linkage between bikunin and H 3 inside pre-a-trypsin inhibitor (Enghild et al. (1991) /. Biol. Chem. 266, 747-751).Carbohydrate analysis of the two heavy chains isolated by ion-exchange chromatography suggests the presence of complex-type ./V-glycans in both H! and H 2 and that of O-glycans in H 2 . HI is eluted from Con-A Sepharose by cc-methylmannoside, in agreement with the existence of at least one biantennary glycan chain. In contrast, H 2 remains strongly bound to this support when submitted to the same conditions. Therefore this binding does not depend on carbohydrates.The capacity of H 2 to develop such interactions is discussed with regard to the unusual bindings likely to exist between the different peptide chains constituting ITI. Die schweren Ketten des Inter-a-Trypsininhibitors: Isolierung und Identifizierung durch Elektrophorese und Teilsequenzierung. Unterschiedliche Reaktivitäten gegenüber Concanavalin A.Zusammenfassung: Der Inter-a-Trypsininhibitor (ITI) ist ein komplexes Protein, das aus einer leichten Kette, dem sogenannten Bikunin besteht sowie aus zwei schweren Ketten mit den apparenten M r -Werten 96000 und 86000 (in der SDS/PAGE unter nicht reduzierenden Bedingungen). Durch Sequenzanalyse konnten wir diese beiden Komponenten klar als HI und H 2 identifizieren.Wir zeigen, daß sowohl alkalische Behandlung (50mM NaOH 5 min bei Raumtemperatur) wie Chondroitase-Verdauung zur Dissoziation von ITI führen. Die für die Alkalibehandlung verwendeten Bedingungen wurden bereits für die Spaltung der kovalenten Bindung zwischen Bikunin und H 3 im Prä-atrypsininhibitor mitgeteilt (Enghild et al. (1991) /. Biol. Chem. 266, 747-751).

Conditions for unblocking reversible chemical modifications such as maleylation or citraconylation 'in situ' at the N-terminus of proteins after transfer of proteins to immobilon membranes from SDS-PAGE are described. Demaleylation or decitraconylation occurred at 55 degrees C in 70% formic acid (pH 1.50) during 60 min. During the unblocking reaction, Coomassie blue dye was completely removed, resulting in superior high performance liquid chromatographic separation of phenylthiohydantoin-amino acid (PTH-AA) after Edman degradation (automatic gas phase sequencer). The protein fixed on the matrix after demaleylation and removal of Coomassie blue was not degraded. The possible cleavage at the aspartyl-prolyl peptide bonds was considered, but no side reaction was observed. Furthermore, the incubation time in 70% formic acid at 55 degrees C could be reduced to 10 min in the absence of maleylation of the starting material, and this was suitable for the removal of Coomassie blue and the quantification of phenylthiolhydantoin-amino acids (PTH-AAs) by HPLC. The yield from the starting protein through SDS-PAGE, blotting, and Edman degradation to quantitative analysis of PTH-aminoacid(s) by HPLC was established.