Arnold Lehmann

Improved automated solid-phase microsequencing of peptides using DABITC

Abstract Attachment studies with EDC (1-ethyl-3-dimethylaminopropyl carbodiimide) have revealed that peptides, ranging from 4 to 31 residues, can be linked to aminopropyl glass with satisfactory yield, if optimal conditions are obeyed. The attachment yields are, however, significantly lower with peptides possessing carboxyl terminal lysine. High-sensitivity solid-phase sequencing of immobilized peptides can be performed if DABITC (4- N, N -dimethylaminoazobenzene 4′-isothiocyanate), an Edman reagent with a covalently linked chromophore, is used in conjunction with phenyl isothiocyanate. Technical improvements have been introduced by utilization of intermittent nitrogen flushes, pumping of the DABITC reagent in a stepwise manner, and incorporation of an automated conversion device based on instant aequous dilution of the trifluoroacetic acid effluent. Solid-phase microsequencing constitutes a method of general applicability for peptides in the 1- to 10-nmol range, with up to 30–35 identifiable degradation steps.

Analysis of demethylaminoazobenzene-thiohydantoins of amino acid by high-pressure liquid chromatography

Abstract Dimethylaminoazobenzene-thiohydantoins of amino acid can be quantitatively analyzed by high-pressure liquid chromatography at picomole level. As little as 5 to 10 pmol of dimethylaminoazobenzene-thiohydantoins of amino acid can easily be detected in the visible region (436 nm) against a stable baseline. Three amino acid pairs, namely glutamine and threonine, methionine and proline, and leucine and isoleucine, have not yet been separated. This new technique provides a sensitive and efficient tool for measuring the recovery of amino terminal amino acids using the dimethylaminoazobenzene-isothiocyanate method and the repetitive yield of sequence determination using the dimethylaminoazobenzene-isothiocyanate phenylisothiocyanate double-coupling method.

Metaphosphate and aerobic CO2 in Chlorella

A striking parellelism between the metaphosphate and the “aerobic CO,” of Chlorella has been observed. If the photolyte [ 1 ] of photosynthesis is built up in Chlorella, the first intermediate is the “aerobic CO,“. It is called “aerobic”, because the fixation of this CO, requires not only CO, but also 0,. The equilibrium of the futation of the aerobic CO, is reached at 20° in 30 minutes. At the same rate the aerobic CO, dissociates if the CO, or the 0, is removed from the gas mixture in the presence of which the aerobic CO, was formed. We have discovered that the highly polymeric metaphosphate of Chlorella increases, when the “aerobic CO,” is formed and that the metaphosphate decreases, when the “aerobic CO,” dissociates on the removal of the CO, or the 0,.

Dialysierte Chlorella, ein neues Versuchsmaterial zur Untersuchung der Photosynthese

Als neues Versuchsmaterial der Photosynthese wird dialysierte Chlorella beschrieben. Mit Hilfe der dialysierten Chlorella wurden zwei bisher unbekannte stöchiometrische Lichtreaktionen entdeckt, die Abspaltung von Kohlensäure aus der aeroben Kohlensäure, sowie die Umwandlung der aeroben Kohlensäuren in den Photolyten.

The use of Dabitc in Automated Solid-Phase Sequencing

The critical factor limiting detailed chemical characterization of physiological important proteins and peptides in the past decade has often been the availability of pure material. Consequently, there is a tendency in protein chemical technology including sequencing towards more sensitive methods with the aim to extend the range of structural studies to these scarce molecules. Although radioactive labelling of the Edman reagent increases the sensitivity range by an order of magnitude, this method is often hampered by the insufficient purity and specific activity of the commercially available compound and the rather cumbersome identification procedure. A different approach has been used by Chang et al. (1) by incorporation of a high absorptive chromophore — an azo-group — into the Edman reagent. The resulting DABITC reagent leads to colored thiohydantoins whose detection limit approaches 5 picomoles permitting microsequencing. The reagent, however, does not yield clean-cut degradation steps unless used in conjunction with PITC in a double coupling procedure (2). Adaptation of this microsequencing method to the solid-phase technique has been successfully attempted (3–5). Here we describe improvements of this technique with respect to peptide attachment, programming of the sequencer and automated conversion.

Chlorophyll catalysis and Einstein's photochemical law in photosynthesis

We have reported [I] that the quantum requirement of the splitting of the photolyte is 1 and [2] that the quantum requirement of the splitting plus resynthesis of the photolyte is 3. This agrees with Einstein’s photochemical law and with thermodynamics. Yet our results were not accepted by the late James Franck or by Melvin Calvin, who reported much higher and erratic quantum requirements. The discrepancies were so enormous that they baffled everyone concerned with photosynthesis. An explanation for the discrepancies is now offered. Photosyntehsis is a chlorophyll catalysis. There are two states of the chlorophyll : chlorophyll combined with carbonic acid (relation 1: 1) and free chlorophyll. Only light absorbed by the combined chlorophyll is the photolyte, whereas light absorbed by the free chlorophyll is lost for photosynthesis. The quantum requirement of photosynthesis can be determined only when the photolyte is known and when it is measured during photosynthesis. Then only light absorbed by the photolyte is introduced into the calculation and light absorbed by the free chlorophyll is ignored, using the equation:

Mit Kohlensäure Verbundenes und Freies Chlorophyll.

Beweis, daß die Funktion des Chlorophylls bei der Photosynthese nicht nur die Absorption des Lichts ist, sondern auch die Umwandlung der Lichtenergie in chemische Energie. Chlorophyll ist also auch der chemische Katalysator der Photosynthese. Der Beweis wurde erbracht durch Messung des verbundenen und des freien Chlorophylls während der Photosynthese sowie der O2-Entwicklung aus dem verbundenen Chlorophyll, deren Quantenausbeute = 1 ist und aus dem freien Chlorophyll, deren Quantenausbeute = 0 ist.

BERICHT Chemie der Photosynthese

Es wird bewiesen, daß die Lichtreaktion der Photosynthese keine Wasserphotolyse, sondern eine Kohlensäurephotolyse ist. Die durch Licht nicht spaltbare Kohlensäure wird dabei durch die Energie der Atmung, unter anderem durch Phosphorylierungen, in durch Licht spaltbare Kohlensäure umgewandelt.