The molecular mechanism of the neutral-to-base transition of human serum albumin. Acid/base titration and proton nuclear magnetic resonance studies on a large peptic and a large tryptic fragment of albumin.

In order to obtain a better understanding of the neutral-to-base (N-B) transition of human serum albumin, we performed acid/base titration experiments and 500-MHz 1H NMR experiments on albumin and on a large peptic (residues 1-387) and large tryptic (residues 198-585) fragment of albumin. The acid/base titration experiments revealed that Ca2+ ions induce a downward pK shift of several histidine residues of the peptic (P46) fragment and of albumin. By contrast, Ca2+ has very little influence on the pK of histidine residues of the tryptic (T45) fragment. In albumin, the pH-dependent His C-2 proton resonances, observed with 1H NMR experiments, have been allotted the numbers 1-17. It proved possible to locate these resonances in the P46 and the T45 fragments. A correspondence was found between the number of histidines detected by the acid/base titration and by the 1H NMR experiments. The results of the experiments lead us to conclude that in domain 1 at least the histidines corresponding to the His C-2 proton resonances 1-5 play a dominant role in the N-B transition. The Cu2+-binding histidine residue 3 (resonance 8) of the albumin molecule is not involved in the N-B transition. In addition, we were able to assign His C-2 proton resonance 9 to histidine 464 of the albumin molecule. The role of the N-B transition in the transport and cellular uptake mechanisms of endogenous and exogenous compounds is discussed.

A proton nuclear magnetic resonance study of human serum albumin in the neutral pH region.

By means of 500 MHz 1H-NMR spectroscopy detailed information has been obtained about the proton titration behaviour of the histidine residues of human serum albumin under various experimental conditions. The approximate number of His C2 protons resolved varied from 11 at pH* 9.0 to 16 at pH* 5.5. The latter is the same as the number of histidine residues reported to be present in the molecule. The symbol pH* stands for the meter readings uncorrected for the deuterium isotope effect. Whereas some of the NMR signals remained visible over the entire pH* range examined (5-9), others, having pH*-dependent resonance intensities, were observed in part of the pH* range only. The latter effect is indicative of an alteration in the histidine environment in the course of pH* titration. It is thought that the so-called N-B conformational transition, occurring in the neutral pH region, is responsible for this alteration. The spectral effects observed upon the addition of Ca2+ can be interpreted in terms of a downward pK* shift for a number of histidine residues and a concomitant downward shift in the midpoint pH* of the N-B transition. This corroborates the results of previous investigations on this matter.

Derivatives of methanopterin, a coenzyme involved in methanogenesis.

Degradational studies of methanopterin, a coenzyme involved in methanogenesis, are reported. The results of these studies are in full accordance with the proposed structure of methanopterin as N-[1'-(2''-amino-4''-hydroxy-7'' -methyl-6''-pteridinyl)ethyl]-4-[2', 3', 4', 5'-tetrahydroxypent-1'-yl(5'-1'' )O-alpha-ribofuranosyl-5''-phosphoric acid] aniline in which the phosphate group is esterified with alpha-hydroxyglutaric acid. Acid hydrolysis of methanopterin cleaved the 5'----1'' glycosidic bond and yielded a 'hydrolytic product' which was identified as N-[1'-(2''-amino-4''-hydroxy-7'' -methyl-6''-pteridinyl)ethyl]-4-[2', 3', 4', 5'-tetrahydroxypent-1'-yl]aniline. Alkaline permanganate oxidation of methanopterin yielded 7-methylpterin-6-carboxylic acid. Catalytic (or enzymatic) hydrogenation of methanopterin gave a mixture of 6-ethyl-7-methyl-7,8-dihydropterin, 6-ethyl-7-methylpterin and a third compound, named methaniline which was identified as 4-[2', 3', 4', 5'-tetrahydroxypent-1'-yl(5'----1'')O-alpha -ribofuranosyl-5''-phosphoric acid]aniline, in which the phosphate group is esterified with alpha-hydroxyglutaric acid. Methanosarcina barkeri contains a closely related coenzyme called sarcinapterin, which was identified as a L-glutamyl derivative of methanopterin, where the glutamate moiety is attached to the alpha-carboxylic acid group of the alpha-hydroxyglutaric acid moiety of methanopterin via an amide linkage.