Highly Diastereoselective Synthesis of (2′S)-[2′-2H]-2′-Deoxyribonucleosides from the Corresponding Ribonucleosides

Abstract

The four (2′S)-[2′-²H]-2′-deoxynucleosides (>90 atom % ²H), were synthesized from the corresponding ribonucleosides involving six steps of reactions, i.e., oxidation of their 2′-hydroxyl group, stereoselective reductive deuteration of the resulting 2′-ketonucleoside intermediates with NaB²H₄ in EtOH-H₂O or EtOH, triflation, bromination with LiBr, highly stereoselective Bu₃SnH-Et₃B reduction of the resulting bromide, and, finally, unmasking.     

Transport, degradation and cell wall-integration of XXFGol, a growth-regulating nonasaccharide of xyloglucan, in pea stems

When [glucitol-³H]XXFGol (a NaB³H₄-reduced xyloglucan nonasaccharide) was applied to excised shoots of pea (Pisum sativum L. cv. Progress) at the base of the epicotyl, it inhibited growth in the elongation zone, 4–5 cm distal. Experiments were conducted to discover whether such ³H-oligosaccharides are translocated intact over this distance, or whether an intercellular second messenger would have to be postulated. After 24 h, ³H from [glucitol-³H]XXFGol and [glucitol-³H]XXXGol showed U-shaped distributions, with most ³H at the base and apex of the stem. Radioactivity from [fucosyl-³H]XXFG and [xylosyl-³H]XXFG also moved acropetally, but did not concentrate at the apex, possibly owing to removal from the transpiration stream of fucose and xylose formed by partial hydrolysis of XXFG en route. When 10⁻⁷ M [glucitol-³H]XXFGol was supplied, about 14 fmol ·  seedling^–1 of apparently intact [³H]XXFGol was extractable from the elongation zone after 24 h. Larger amounts of degradation products were extractable (including free [³H]glucitol) and some wall-bound ³H-hemicellulose was present (presumably formed by the oligosaccharides acting as acceptor substrates for transglycosylation). We conclude that biologically active oligosaccharides of xyloglucan can move through the stem acropetally and that they are maintained at low steady-state concentrations by both hydrolysis and transglycosylation. Received: 1 April 1997 / Accepted: 28 May 1997     

Synthesis and biological activity of [14a-3H]cryptopleurine

[14a-³H]Cryptopleurine was chemically synthesized from the perchlorate salt of 9,11,12,13,14,15-hexahydro-2,3,6-trimethoxyphenanthro(9,10-b)quinolizidinium by reduction with NaB³H₄. The [³H]cryptopleurine was recrystallized from acetone and further purified by chromatography through alumina using benzene as the eluting solvent. Both infrared and ultraviolet spectra of the labeled product were identical to those obtained using either the natural compound or the unlabeled synthetic compound. Thin-layer analysis on various solid supports using several different eluting solvents gave only one radioactive spot with a specific activity of 1438 Ci/mol, which in all cases cochromatographed with the natural sample. The [³H]cryptopleurine was also identical to the unlabeled compound in that it bound strongly to polyribosomes. 80 S ribosomes, and 40 S ribosomal subunits, all isolated from yeast. Binding was less strong using either 60 S ribosomal subunits or Escherichia coli ribosomes.     

Interaction of homo-aza-steroidal ester of [p-[bis-(2-chloroethyl) amino]phenyl]acetic acid (ASE) with DNA of Ehrlich ascites tumor cells

A cytostatic, homo-aza-steroidal ester of [p-[bis-(2-chloroethyl) amino]phenyl]acetic acid (ASE) was reduced with NaB³H₄ and [³H]ASE-treated DNA prepared in vitro. We found that: (1) ASE reacts preferentially with purines; (2) ASE decreases the thermal stability of the double helix upon binding to DNA; (3) [³H]ASE binding sites are clustered along the DNA molecules; (4) ASE binding sites probably represent oligo- or polypurine sequences.     

Synthesis and separation of tritium-labeled intermediates of the shikimate pathway

[5-³H]Shikimate (sp radioact 2000 Ci/mol) has been synthesized by reduction of the methyl ester of 5-dehydroshikimate with NaB³H₄ and subsequent hydrolysis of the ester group (M. M. Leduc, P. M. Dansette, and R. G. Azerad (1970) Eur. J. Biochem.15, 428–435). The [5-³H]shikimate has been converted enzymatically to [5-³H]chorismate and [5-³H]prephenate of similar high specific radioactivity by using a cell-free extract of Aerobacter aerogenes 62-1. In addition, a chromatograhic procedure, which utilizes polyethyleneimine-cellulose thin-layer chromatograms, has been developed for the separation of intermediates along the shikimate pathway between shikimate and hydroxyphenylpyruvate or phenylpyruvate. Since the method allows quantitative measurement of tritium-labeled intermediates, it provides the basis for sensitive radioassays of the individual enzymes and allows study of the reaction flux along the overall pathway. The same intermediates can be separated on a large scale by use of a column of DEAE-Sephacel.     

Stereochemical action of mouse brain glutamate decarboxylase

4-[4-²H]Aminobutyrate was prepared by incubation in ²H₂O of glutamate with a partially purified glutamate decarboxylase from mouse brain. The 4R configuration was assigned to the compound on the basis of ¹H nmr analysis of the ω-camphanoylamide of its methyl ester in the presence of Eu(dpm)₃. Moreover 4-[4(S)4-³H,U-¹⁴C]aminobutyrate was shown to be formed from [2(S)2-³H,U-¹⁴C]glutamate by the same enzyme fraction. It is therefore demonstrated that glutamate decarboxylation catalyzed by this enzyme preparation occurs with retention of configuration.     

Effect of Photoperiod on Metabolism of [H] Gibberellins A(1), 3-epi-A(1), and A(20) in Agrostemma githago L

To determine whether daylength influences the rate of metabolism of gibberellins (GAs) in the long-day (LD) rosette plant Agrostemma githago L., [³H]GA₂₀ and [³H]GA₁ were applied under short day (SD) and LD. Both were metabolized faster under LD than under SD. [³H]GA₂₀ was metabolized to a compound chromatographically identical to 3-epi-GA₁. [³H]GA₁ was metabolized to two acidic compounds, the major metabolite having chromatographic properties similar to, but not identical with GA₈. [³H]3-epi-GA₁ applied to plants under LD was metabolized much more slowly than was [³H]GA₁, and formed a very polar metabolite which did not partition into ethyl acetate at pH 2.5. Very polar metabolites were also formed after the feeds of [³H]GA₂₀ and [³H]GA₁. It was not possible to characterize these very polar compounds further because of their apparent instability. The results obtained suggest that in Agrostemma GA₂₀ is the precursor of 3-epi-GA₁, but there is at present no evidence indicating the precursor of GA₁.     

Stereochemistry of Decarboxylation Reactions Catalyzed by a Constitutive Aromatic l-Amino Acid Decarboxylase

The stereochemistry of the decarboxylation reaction catalyzed by an aromatic l-amino acid decarboxylase, purified from Micrococcus percitreus, was studied using stereospecifically deuterium labelled phenylalanine (Phe). The ¹H NMR spectrum of [1,2-²H₂]-β-phenethylamine enzymatically derived from (2S, 3R)-[3-²H]-Phe in ²H₂O was compared with that of [1-²H]-β-phenethylamine from unlabelled Phe in ²H₂O. The results clearly indicate that the decarboxylation reaction of this enzyme proceeds exclusively through a course in which the configuration at C-2 of Phe is retained.     

Formation of 3H2O from [2-3H]- and [4-3H]estradiol by rat uteri in vitro: Possible role of peroxidase

The mitochondrial fraction of diethylstilbestrol-treated rat uteri, known to contain an estrogen-induced peroxidase, was able to catalyze the release of ³H₂O from either [2-³H]- or [4-³H]estradiol. Hydrogen peroxide added to this system increased the yield of ³H₂O but had no effect on mitochondrial preparations from ovariectomized rat uteri having only very low peroxidase activity. The reaction was inhibited by catalase and also occurred with lactoperoxidase in the presence of H₂O₂ but 2-hydroxyestradiol was not detected in any of these experiments. Under similar conditions, tyrosinase catalyzed the formation of the catechol estrogen with loss of ³H from [2-³H]- or [2,4,6,7-³H]- but not [4-³H]- or [6,7-³H]estradiol. It is proposed that the formation of ³H₂O from ³H-labeled estradiol in the estrogen-treated rat uterus may occur by a peroxidative mechanism which does not necessarily result in hydroxylation of the steroid.     

Mechanistic and metabolic studies of sterol 24,25-double bond reduction in Manduca sexta

Larvae of Manduca sexta were used to obtain a cell-free sterol 24,25-reductase. From the midgut of fifth instar larvae fed a mixture of sitosterol and campesterol a microsome-bound 24,25-sterol reductase was prepared that transformed desmosterol (K_m, 3 μM), lanosterol (K_m, 18 μM), and cycloartenol (K_m, 33 μM), to cholesterol, 24,25-dihydrolanosterol, and cycloartanol, respectively. With desmosterol as substrate, the microsome-bound enzyme was found to incorporate tritium into cholesterol from 4S-tritium labelled NADPH. [24-²H]lanosterol was transformed by larvae to [24-²H]24,25-dihydrolanosterol (structure confirmed by mass spectroscopy (MS) and ¹H-nuclear magnetic resonance spectroscopy. A rationally designed inhibitor of 24,25-reductase activity, 24(R,S),25-epimino-lanosterol (IL), was assayed and found to be inhibitory with an I₅₀ of 2 μM. IL was supplemented in the diet of M. sexta with either sitosterol or stigmasterol and found to inhibit development (I₅₀ 60 ppm). The major sterol which accumulated in the IL-treated larvae was desmosterol, confirming the site of inhibition was reduction of the 24,25-bond. IL was converted to [2-³H]IL when fed to the larvae. [2-³H]lanosterol was recovered from fifth instar larvae and its structure confirmed by MS and radiochemical techniques. © 1996 Wiley-Liss, Inc.     

Simultaneous determination of stable isotopically labelled l-histidine and urocanic acid in human plasma by stable isotope dilution mass spectrometry

A capillary gas chromatographic—mass spectrometric method for the simultaneous determination of stable isotopically labelled l-histidine (l-[3,3-²H₂,1′,3′-¹⁵N₂]histidine, l-His-[M + 4]) and urocanic acid ([3-²H,1′,3′-¹⁵N₂]urocanic acid, UA-[M + 3]) in human plasma was developed using dl-[2,3,3,5′-²H₄,2′-¹³C,1′,3′-¹⁵N₂]histidine (dl-His-[M + 7]) and [2,3,5′-²H₃,2′-¹³C,1′,3′-¹⁵N₂]urocanic acid (UA-[M + 6]) as internal standards. l-Histidine and urocanic acid were derivatized to ^αN-(trifluoroacetyl)-^imN-(ethoxycarbonyl)-l-histidine n-butyl ester and ^imN-(ethoxycarbonyl)urocanic acid n-butyl ester. Quantification was carried out by selected ion monitoring of the molecular ions of the respective derivatives of l-His-[M + 4], dl-His-[M + 7], UA-[M + 3] and UA-[M + 6]. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring plasma concentrations of l-His-[M + 4] and UA-[M + 3] following administration of trace amounts of l-His-[M + 4] to humans.     

Charcoal-catalyzed transfer of tritium into 3H2O from regiospecifically-labeled 2-hydroxyestradiol in the presence of thiols

Charcoal was found to catalyze the release of ³H₂O from [1-³H]2-hydroxyestradiol-17β ([1-³H]2-OHE₂) or [4-³H]2-hydroxy-estradiol-17β ([4-³H]2-OHE₂) and this effect was shown to occur in the presence of glutathione or other thiols and to depend on the concentration of free steroid. The radiometric assay for measuring the formation of ³H₂O was not affected significantly by subsequent treatment of the incubation mixture with charcoal if the ratio of steroid to tissue (rat brain or liver microsomes) was low and only initial rates of ³H release were measured. 2-Hydroxyestradiol did not show the charcoal effect in the presence of tyrosinase, either when it was generated from its parent estrogen or added to the enzyme. The formation of ³H₂O from [4-³H]2-OHE₂ in the presence of glutathione was inhibited by ascorbic acid but the addition of dextran or albumin did not protect the catechol estrogen from the charcoal-catalyzed loss of tritium. The reaction with glutathione and charcoal occurred even at 4°C but other adsorbants such as alumina, silica or hydroxylapatite were without effect.     

Biosynthesis of vitamin E and of the plastoquinones in chloroplasts: Steric course of the decarboxylation

Samples of (3R)- and (3S)-4′hydroxyphenyl[3-²H₁, 3-³H]pyruvate were prepared by taking advantage of the known stereospecificity of phenylpyruvate keto-enol isomerase (tautomerase). 4′-Hydroxyphenyl[3-¹⁴C]pyruvate was obtained by the action of l-amino acid oxidase on dl-[3-¹⁴C]tyrosine, whereas a simple base-catalyzed exchange procedure yielded samples of 4′-hydroxyphenyl[3-³H]- and 4′-hydroxyphenyl[3-²H₂]pyruvate. All labeled samples were converted in situ into the corresponding homogentisic acids on 4′-hydroxyphenyl-pyruvate dioxygenase that is known to catalyze the migration of the acetate side chain with retention of configuration. The isolated doubly labeled homogentisic acids were incubated with chloroplasts from Raphanus sativus cv. saxa Treib, and from the lipophilic products a fraction containing inter alia tocopherol, tocoquinone, and plastoquinone was obtained by chromatographic procedures. The incorporation of radioactivity was between 0.5 and 11% based on homogentisate. Reductive acetylation of the quinones yielded crystalline diacetylhydroquinones, which were submitted to Kuhn-Roth degradation. The radioactive acetate samples thus obtained were analyzed for chirality by an enzymatic procedure previously published. (2R)-[2-²H₁, 2-³H]Homogentisate gave mainly (S)-acetate, whereas (2S)-[2-²H₁, 2-³H]homogentisate was converted mainly into (R)-acetate. It is concluded that the decarboxylation of the side chain occurred with stereochemical retention during the biosynthetic process.     

Lack of influence of photoperiod on the metabolism of gibberellin A20 in Salix pentandra

The influence of photoperiod on the metabolism of GA₂₀ in Salix pentandra was studied by feeding [³H]-GA₂₀ to seedlings which had been grown previously under long day (LD) or short day (SD) conditions. After 48 h in LD or SD, metabolites were separated on sequential, silica gel partition columns and reversed-phase C₁₈ HPLC. The principal metabolite co-chromatographed with [³H]-GA₁ and this conversion was confirmed by feeding [²H]-GA₂₀, which was converted to [²H]-GA₁ as identified by gas chromatography-selected ion monitoring. Chromatographic evidence also indicated the minor conversion of [³H]-GA₂₀ to [³H]-GA₈ (via [³H]-GA₁) and trace conversion to [³H]-GA₂₉ (GAs A_1.8,20.29 are native in Salix). Ethyl acetate-insoluble [³H] metabolites were formed and could be cleaved by cellulase to release putative [³H]-GA₂₀ and [³H]-GA₁ suggesting the conversion to glucosyl conjugates of these GAs. Metabolism of [³H]-GA₂₀ was slightly more rapid in plants previously grown under LD than SD, an effect which reflected the generally increased shoot growth under LD. However, altering the photoperiod after [³H]-GA₂₀ addition had only a slight effect on the metabolism of [³H]-GA₂₀ in Salix seedlings. This indicates that the conversion of GA₂₀ to GA₁ is not a controlling step in the photoperiodic regulation of growth cessation in Salix.     

Biosynthesis of Violacein: Origins of Hydrogen,Nitrogen and Oxygen Atoms in the 2-Pyrrolidone Nucleus

The biosynthetic origins of the hydrogen, nitrogen and oxygen atoms in the pyrrolidone ring of violacein'were established by an anaylses of the ¹H, ¹³C NMR and MS spectra of its isotope-enriched metabolites. Feeding experiments of [2-²H] and [3-²H₂]tryptophans have revealed that the hydrogen in the pyrrolidone ring was derived from the methylene protons at the 3-position of tryptophan. The stereochemical fate of the prochiral hydrogens was determined to be in the retention of the pro-S hydrogen by these feeding experiments using [3R-²H] and [3S”-²H]tryptophans. The incorporation experiment of [α-¹⁵N]tryptophan demonstrated that the nitrogen atom in the ring originated from the α-amino group of tryptophan. The incorporation experiment of ¹⁸O₂ gas verified that all the oxygen atoms of violacein were derived from the molecular oxygen.     

Dormancy in somatic embryos and seeds ofVitis: changes in endogenous abscisic acid during embryogeny and germination

Abscisic acid (ABA) in extracts of somatic embryos and seeds of Gloryvine (Vitis vinifera L.xV. rupestris Scheele) was measured by gas chromatography-mass spectrometry-selected ion monitoring using deuterated ABA, (±)-[C-3Me-²H₃]ABA, ([²H₃]ABA) as internal standard. The ABA content increased rapidly during embryogeny (0.035 ng/embryo at the globular stage to 0.22 ng/embryo at the mature stage). The level of ABA in the tissues of somatic embryos, expressed in ng/mg dry weight, decreased from the globular stage (0.76 ng/mg) to the mature stage (0.25 ng/mg). Chilling (4° C) induced normal germination of seeds and mature somatic embryos and precocious germination of globular, heart-shaped and torpedoshaped somatic embryos. In all cases chilling led to a marked reduction in endogenous ABA. Exogenous (±)-ABA inhibited the germination of chilled somatic embryos.Abbreviations ABA abscisic acid - [²H₃]ABA (±)-[C-3Me-²H₃]-abscisic acid - BHT 2,6-di-t-butyl-4-methylphenol - GC-MS gas chromatography-mass spectrometry - Me-ABA and Me-[²H₃]ABA methyl esters of ABA and [²H₃]ABA, respectively - SIM selected ion monitoring     

Chemical modification of an essential lysine at the active site of enoyl-CoA reductase in fatty acid synthetase

Fatty acid synthetase from goose uropygial gland was inactivated by treatment with pyridoxal 5′-phosphate. Malonyl-CoA and acetyl-CoA did not protect the enzyme whereas NADPH provided about 70% protection against this inactivation. 2′-Monophospho-ADP-ribose was nearly as effective as NADPH while 2′-AMP, 5′-AMP, ADP-ribose, and NADH were ineffective suggesting that pyridoxal 5′-phosphate modified a group that interacts with the 5′-pyrophosphoryl group of NADPH and that the 2′-phosphate is necessary for the binding of the coenzyme to the enzyme. Of the seven component activities catalyzed by fatty acid synthetase only the enoyl-CoA reductase activity was inhibited. Inactivation of both the overall activity and enoyl-CoA reductase of fatty acid synthetase by this compound was reversed by dialysis or dilution but not after reduction with NaBH₄. The modified protein showed a characteristic Schiff base absorption (maximum at 425 nm) that disappeared on reduction with NaBH₄ resulting in a new absorption spectrum with a maximum at 325 nm. After reduction the protein showed a fluorescence spectrum with a maximum at 394 nm. Reduction of pyridoxal phosphate-treated protein with NaB³H₄ resulted in incorporation of ³H into the protein and paper chromatography of the acid hydrolysate of the modified protein showed only one fluorescent spot which was labeled and ninhydrin positive and had an R_f identical to that of authentic N⁶-pyridoxyllysine. When [4-³H]pyridoxal phosphate was used all of the ³H, incorporated into the protein, was found in pyridoxyllysine. All of these results strongly suggest that pyridoxal phosphate inhibited fatty acid synthetase by forming a Schiff base with the ?-amino group of lysine in the enoyl-CoA reductase domain of the enzyme. The number of lysine residues modified was estimated with [4-³H]pyridoxal-5′-phosphate/NaBH₄ and by pyridoxal-5′-phosphate/NaB³H₄. Scatchard analysis showed that modification of two lysine residues per subunit resulted in complete inactivation of the overall activity and enoyl-CoA reductase of fatty acid synthetase. NADPH prevented the inactivation of the enzyme by protecting one of these two lysine residues from modification. The present results are consistent with the hypothesis that each subunit of the enzyme contains an enoyl-CoA reductase domain in which a lysine residue, at or near the active site, interacts with NADPH.     

Characterization of binding of [3H]PD 128907, A selective Dopamine D3 receptor agonist ligand,to CHO-K1 cells

PD 128907 [4a R, 10 b R-(+)-trans- 3, 4, 4a, 10 b - tetrahydro - 4- n-propy12 H,5H-[1] benzopyrano[4,3-b]1,4-oxazin-9-ol.], a selective dopamine (DA) D3 receptor agonist ligand exhibits about a 1000-fold selectivity for human D3 receptors (K_i, 1 nM) versus human D₂ receptors (K_i, 1183 nM) and a 10000-fold selectivity versus human D₄ receptors (K_i, 7000 nM) using [³H]spiperone as the radioligand in CHO-K1-cells. Studies with [³H]PD 128907, showed saturable, high affinity binding to human D₃ receptors expressed in CHO-K1 cells (CHO-K1-D₃) with an equilibrium dissociation constant (K_d) of 0.99 nM and a binding density (B_max) of 475 fmol/mg protein. Under the same conditions, there was no significant specific binding in CHO-K1-cells expressing human D₂ receptors (CHO-K1-D₂). The rank order of potency for inhibition of [³H]PD 128907 binding with reference DA agents was consistent with reported values for D₃ receptors. These results indicate that [³H]PD 128907 is a new, highly selective D₃ receptor ligand with high specific activity, high specific binding and low non-specific binding and therefore should be useful for further characterizing the DA D₃ receptors.     

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates: isotopic discrimination between hydrogen and deuterium

Summary The discrimination between the isotopes of hydrogen in the reaction catalyzed by yeast phosphoglucoisomerase is examined by NMR, as well as by spectrofluorometric or radioisotopic methods. The monodirectional conversion of D-glucose 6-phosphate to D-fructose 6-phosphate displays a lower maximal velocity with D-[2-²H]glucose 6-phosphate than unlabelled D-glucose 6-phosphate, with little difference in the affinity of the enzyme for these two substrates. About 72% of the deuterium located on the C₂ of D-[1-¹³C,2-²H]glucose 6-phosphate is transferred intramolecularly to the C₁ of D-[1-¹³C,1-²H]fructose 6-phosphate. The velocity of the monodirectional conversion of D-[U-¹⁴C]glucose 6-phosphate (or D-[2-³H]glucose 6-phosphate) to D-fructose 6-phosphate is virtually identical in H₂O and D₂O, respectively, but is four times lower with the tritiated than ¹⁴C-labelled ester. In the monodirectional reaction, the intramolecular transfer from the C₂ of D-[2-³H]glucose 6-phosphate is higher in the presence of D₂O than H₂O. Whereas prolonged exposure of D-[1-¹³C]glucose 6-phosphate to D₂O, in the presence of phosphoglucoisomerase, leads to the formation of both D-[1-¹³C,2-²H]glucose 6-phosphate and D-[1-¹³C,1-²H]fructose 6-phosphate, no sizeable incorporation of deuterium from D₂O on the C₁ of D-[1-¹³C]fructose 1,6-bisphosphate is observed when the monodirectional conversion of D-[1-¹³C]glucose 6-phosphate occurs in the concomitant presence of phosphoglucoisomerase and phosphofructokinase. The latter finding contrasts with the incorporation of hydrogen from ¹H₂O or tritium from ³H₂O in the monodirectional conversion of D-[2-³H]glucose 6-phosphate and unlabelled D-glucose 6-phosphate, respectively, to their corresponding ketohexose esters.     

Preparation, properties, and uses of two fluorogenic substrates for the detection of 5'-(venom) and 3'-(spleen) nucleotide phosphodiesterases

A new method for ³H-labeling of native collagen and a specific microassay for collagenase activity are presented. Acid-soluble type I collagen derived from rat tail tendons was reacted with pyridoxal phosphate and then reduced with NaB³H₄ to yield [³H]collagen with a specific activity of more than 10 μCi/mg. With respect to rate of hydrolysis, trypsin susceptibility, and gelling properties this collagen compares favorably with biosynthetically labeled preparations. It was shown that chemical labeling procedures such as this, or N-acetylation with acetic anhydride, do not adversely affect properties of collagen which are important for its use as substrate in specific assays. The microassay employs 50-μl [³H]collagen gels (1 mg/ml) dispensed in microtest plates. At 36°C this assay combines rapid rate of hydrolysis with low trypsin susceptibility. As little as 1 ng of clostridial collagenase activity can be measured reproducibly. The high specific activity of the [³H]collagen allowed us to explore microassay conditions employing minute quantities of substrate in solution. These studies indicated that native type I collagen whether labeled or not, is cleaved in the helical region by trypsin at subdenaturation temperatures. It was concluded that, in order to remain specific, collagenase assays with collagen in solution as with collagen in fibrils must be performed at 10–12°C below the denaturation temperature, i.e., at 35–37°C with collagen gels and 27–29°C with collagen in solution.