Urea and plasma protein differ in natural 15N abundance up to 10%. The origin of this difference is the branched nitrogen metabolism in the liver. One main branch is the protein synthesis pathway, the other the urea synthesis pathway. By this branching 15N of precursor amino acids is depleted in urea while it is enriched in protein. With the 15N abundance of precursor amino acids, which may be taken from jejunum tissue, utilization of amino acids in liver metabolism can be calculated from isotope discrimination in either pathway. This was investigated by feeding different proteins to rats. When feeding high quality protein (whey protein) utilization of amino acids in liver metabolism at requirement intake was better than at zero protein intake (> 85% vs. 70%). From this we conclude that the pattern of amino acids available from the metabolic pool at zero protein intake is characterized by an imbalance. This endogenous imbalance can be complemented by exogenous dietary amino acids so that nitrogen excretion may even be smaller than the so-called "obligatory" losses of intakes not exceeding requirement. Thus, the quality of dietary protein is reflected not only by N balance. It also may be quantified by analysis of isotope discrimination in nitrogen metabolism of the liver. In addition, the quality of amino acid pattern available from the metabolic pool is indicated by this method.

The GO binding properties of monomeric (Hb I11 and IV), dimeric, and tetrameric Chironomus haemoglobins as well as of the monomer-analogue valence hybrid (aFmet /Imet /Ideox') of Hb M Iwate were studied and compared with the respective 0, binding properties. All these haemoglobins show hyperbolic 0, binding curves and a Bohr effect. The amplitudes of the 0, Bohr effect curves range from dlog pO,(,,r, = 0.3 (Hb 111) to 2.4 (tetrameric Chironomus haemoglobin).The ligands CO and 0, differ not only in affinity but also with regard to the Bohr effect. The GO affinities of the various Chironomus haemoglobins are 60 to 490 times larger than the 0, affinities and the amplitudes of the CO Bohr effect curves are 1.25 to 8 times smaller than those of the respective 0, Bohr effect curves. I n the case of Hb M Iwate, however, the CO Bohr effect is larger than the 0, Bohr effect by the factor 1.25 (2,3-bisphosphoglycerate-reacted H b M) and 1.4 (stripped Hb M).The ligand-specific differences in the magnitude of the Bohr effect cannot be explained solely by the allosteric interaction connected with the trans-effect of the complex. The differences are discussed in terms of steric influences on the binding geometry of the 6th ligand by protein side chains a t the distal side of the haem group. The titration behaviour of the three titratable histidine residues of H b I11 was studied by proton magnetic resonance (PMR) spectroscopy [3]. Ligation with CO produces a shift of the pK value of the histidine G2. This residue was identified, therefore, as the Bohr proton-binding site. As the PMR measurements required periods of several hours at 30 "C, it was not possible to use 0, as 6th ligand because of autoxidation of the haemoglobin. However, the kind of ligand bound to the 6th position seems to play an important role with respect to the extent of the pH-dependent change of the affinity for, in contrast to the experiments with CO, the PMR studies on ferric and ferrous H b I I I , ligated with Dedicated to Prof. Dr Hans Netter (Institut fiir Physiologische Chemie und Physikochemie der Christian-AlbrechtsUniversitat zu Kiel) on the occasion of his 75th birthday.Abbreviations. Hb I, Hb I11 and Hb IV, monomeric haemoglobins I, I11 and IV from Chironomus thummi thummi; Hb M Iwate, human haemoglobin M Iwate; PMR, proton magnetic resonance. The CO equilibrium studies were extended also to other monomeric, dimeric and tetrameric haemoglobins of Chironomus and to a mutant tetrameric haemoglobin (Hb M Iwate) which is characterized by one or two GO binding sites, respectively [6,7]. CN-MATERIALS AND METHODS HaemoglobinsMonomeric haemoglobin I, 111, and IV fromChironornus thummi thummi were isolated as described in [3].Eur. J. Biochem. 45 (1974)

Thirty-six miniature pigs divided into two groups of 18 animals were fed 15N-labeled milk or yogurt. Polyethylene glycol 4000 was added to the diets as a non-absorbable marker of the liquid phase. Animals were slaughtered 1, 2, 4, 8 and 12 h after meal ingestion, and the gastrointestinal tract was removed and divided into 10 parts. Polyethylene glycol, total nitrogen and 15N enrichment were measured in the digesta. Both the intestinal delivery of the liquid phase and the nitrogenous fraction of the chyme were delayed more in pigs fed yogurt than in pigs fed milk. No stimulatory effect of diet ingestion on endogenous nitrogen secretion was found. Both milk proteins and yogurt proteins were highly digestible: 93% of the exogenous nitrogen disappeared 12 h after feeding. The kinetics of exogenous nitrogen delivery into the intestine was correlated (r = 0.999 for milk and r = 0.974 for yogurt) with that of exogenous nitrogen absorption. These results suggest that milk proteins are rapidly absorbed after they reach the intestine. Gastric emptying is a major factor controlling the kinetics of milk nitrogen absorption.

Milk and yoghurt proteins were I5N-labelled in order to measure the flow rate of exogenous N during digestion in the human intestine. After fasting overnight, sixteen healthy volunteers, each with a naso- A great difficulty in the study of intestinal protein digestion in humans arises from the fact that after ingestion, dietary proteins are mixed with endogenous proteins secreted in the lumen, i.e. gastric, bilio-pancreatic and intestinal secretions. In addition, contrary to fasting conditions where endogenous proteins are continuously secreted into the lumen at a constant level (Gaudichon et al. 1994a), the ingestion of a meal stimulates salivary, gastric and bilio-pancreatic secretions (Alpers, 1987) to a level which depends on the amount and on the nature of the meal and particularly its dietary protein content (Lurie et al. 1973;Girard-Globa et al. 1980;Simon et al. 1983

The monomeric insect (Chironomus thummi thummi) haemoglobins CTT 111 and CTT IV show an alkaline Bohr effect. The amplitude of the Bohr effect curve of CTT IV is about twice as large as that of CTT 111. In particular, at low pH a time-dependent 'slow' decrease in p s o upon cyclic oxygenation/deoxygenation is observed which is larger if dithionite, instead of ascorbate, is the reducing agent. The decrease of p s o (increase in affinity) correlates with the ratio of haem-rotational components exhibiting an increase of the 'myoglobin-like' haemrotational component with high O2 affinity and high stability of the globin-haem complex.The replacement of protohaem IX by mesohaem IX and deuterohaem IX, respectively, causes an increase in O2 affinity following the order: proto < meso < deutero CTT Hbs. The Bohr effect, however, seems not to be affected by these porphyrin side-group substitutions. The O2 affinity is modulated by steric effects due to the substituents in position 2 and 4 via variation of the protein-haem interactions which influence the O2 release.The replacement of iron by cobalt in proto and meso CTT IV leads to an increase of the p.30 by two to three orders of magnitude. Neither central metal nor vinyl replacement affect the Bohr effect.The natural CTT Hbs 111 and IV analyzed for mono-componential kinetic systems exhibit pH-dependent O2 off-rate constants: 300 s-' (at pH 5.6) and 125 s-l (at pH 9.7) for CTT 111, and 550 s-l (at pH 5.4) and 100 s-' (at pH 9.0) for CTT IV. Inflection points and amplitudes of the log koff/pH plots correspond to those obtained from the Bohr effect curves indicating again a larger Bohr effect for CTT IV than for CTT 111. In contrast, the O2 on-rate constants are pH-independent (Icon = 1.15 -1.26 x lo8 M-' s-l). Thus, the Bohr effect is completely controlled by the off-rate constants.Analysis for bi-componential kinetic systems employing the eigenfunction expansion method clearly identifies two kinetic components for proto-IX and deutero-IX CTT Hbs which can be attributed to the two haem-rotational components x and y ( x and y differ due to an 180" rotation of the haem group about the qy-meso axis; y is the The monomeric insect haemoglobins CTT I11 and CTT IV exhibit a Bohr effect [l -31 and therefore serve as simple allosteric model systems [4, 51. Changes in ligation or in pH have no effect on the molecular mass of these haemoglobins which are therefore monomeric under all conditions [2, 61. The pH-dependent ligand afinity is controlled by a single proton (Bohr proton) [2,3,7 -91. NMR titration experiments [7] and histidine assignments based on X-ray structure analysis [lo] provided evidence that the allosteric site in CTT 111 (Bohr proton binding site) is a salt bridge formed by the imidazole group of His-G2 and the C-terminal carboxyhc group of Met-H22. At low pH, this salt bridge stabilizes the tense tertiary structure (t state) characterized by low O2 affinity. At high pH, by dissociation of the Bohr proton, the salt bridge is opened leading to a transition of the terti...