Joachim Otto

Solid-phase Edman degradation of a protein: N-terminal sequence of cytochromec fromCandida krusei

Recently, the quafftitative attachment of cytochrome c from horse heart to the p-phenylene diisothiocyanate (DITC) derivative of 3-aminopropyl glass (APG) as a support for solid-phase Edman degradation was demonstrated [ 1 ]. In the present study, cytochrome c from Candida krusei was used because it has a free a-amino group in contrast to cytochrome c from horse heart which is acetylated at its N.terminus. The C. krusei cytochrome c has been sequenced by Narita and Titani using conventional methods [2]. This protein was degraded by automated solid-phase Edman degradation over 35 steps with an average repetitive yield of 95%. In the course of this degradation we found that Glu in position 22 [2] actually in Gin. The results indicate that the scope of the solid-phase method might be extended to longer N-terminal sequences of proteins by the use of controlled-pore glass (CPG) derivatives as support and, in connection with this, by an optimized degradation cycle.

[24] Chromatography on microbore columns

Publisher Summary The procedures of higher sensitivity are desirable for the purification of peptides resulting from the cleavage of proteins. Compared to other separation techniques, column chromatography has several advantages: it is essentially preparative, well suited for automation, and the most versatile in selectivity. The sensitivity of column chromatography is increased by the use of microbore columns, which involve the use of Sephadex gels or Bio-Gels and Sephadex ion-exchangers. On such columns, peptides can be purified in amounts between 10 and several 100 nmol. The purification procedure is facilitated by the analytical information obtained from nondestructive detectors. Because of the reduced column diameter, the separations are highly reproducible and, owing to their small volume, the work-up of fractions is simplified. Any type of pump may be used in the chromatography that is capable of delivering solvents at constant flow rates between 0.05 and 5.0 ml/hr. The flow rates should be independent of column back pressure in the range of 0-50 psi.

Automatische Säulen-Chromatographie von Peptiden im Nanomolmaßstab

Methods are described for the chromatographic resolution of peptides at the 10–200 nanomole range. Separation is achieved on 0.1×50 cm capillary columns with a sulfonated polystyrene cation-exchanger or with the anion-exchanger QAE-Sephadex. The columns are eluted by suitable gradients of pH and salt at a flowrate of 0.4 ml per hour.By automatic detection with 2,4,6-trinitrobenzene-sulfonic acid peptide elution patterns are produced with 10–50 nanomoles. For preparative chromatography with the same columns, the peptides are detected by monitoring at 280/289 nm resp. 220 nm.The effluent samples from the columns are desalted and further fractionated by gel filtration (Sephadex G 10–G 25) on 0.4×100 cm columns. The isolation of two tryptophane-containing peptides from phosphoglycerate kinase from yeast is demonstrated.ZusammenfassungEs werden Methoden zur automatischen Säulen-Chromatographie von Peptiden in Mengen von 10–200 nMol beschrieben. Die Ionenaustausch-Chromatographie erfolgt in 0,1×50 cm-Capillarsäulen an einem Polystyrol-Sulfonsäure-Kationenaustauscher oder an dem Anionenaustauscher QAE-Sephadex. Die Säulen werden mit geeigneten pH- und Salzgradienten mit einer Flußrate von 0,4 ml/h eluiert.Durch den automatisierten Nachweis mit 2,4,6-Trinitrobenzolsulfonsäure werden aus 10–50 nMol analytische Peptidmuster von Proteinen gewonnen. Zur präparativen Chromatographie an den gleichen Säulen werden die Peptide durch direkte Photometrie bei 280/289 nm bzw. bei 220 nm nachgewiesen.Die Entsalzung und weitere Auftrennung erfolgt durch Gelfiltration in 0,4×100 cm-Säulen mit Sephadex G 10-G 25. Als Beispiel wird die Isolierung zweier tryptophanhaltiger Peptide der Phosphoglyceratkinase aus Hefe demonstriert.

On the Linkages between Chromophore and Protein in Biliproteins, VII

Bilipeptides from all chromophore regions were prepared by trypsin digestion of C-phycoerythrin from Pseudanabaena W 1173 and Phormidium persicinum. Analytical separation and quantitative determination of bilipeptides was achieved by isoelectric focusing, preparative isolation by gel chromatography and ion-exchange chromatography. Amino acid analysis revealed cysteine as the only amino acid common to all chromopeptides. Amino acid sequences were determined by Edman degradation and the dansyl-Edman technique. Sequences are different in all 5 and 6 chromophore regions, respectively. Possible homologies are discussed. A thioether linkage between ring A of the chromophore and cysteine was found in the bilipeptides (as before in biliproteins). A second linkage (serine ester) was found in only one peptide (CM 4.I from Pseudanabaena W 1173). This peptide absorbs as cation at a longer wavelength (559 nm) than the other bilipeptides (542 - 550 nm).