Stereospecificity of sodium borohydride reduction of pig kidney dopa decarboxylase

Sodium boro[3H]hydride reduction of pig kidney 3,4 dihydroxyphenylalanine decarboxylase followed by complete hydrolysis of the enzyme produced epsilon-[3H]pyridoxyllysine. Degradation of this material to 4'-[3H]pyridoxamine and stereochemical analysis with apoaspartate aminotransferase showed that the re side at C-4' of the coenzyme is exposed to solvent. In order to determine the face exposed to the solvent in the external Schiff's base, attempts to trap reaction intermediates were made by reduction with sodium boro [3H]hydride of the holoenzyme in the presence of various substrates or substrate analogs. In all cases, covalently bound radioactive material was found which was identified as epsilon-N-pyridoxyllysine. These results suggest that the internal Schiff's base is in mobile equilibrium with the external Schiff's base and that sodium borohydride reduction displaces this equilibrium, resulting in complete reduction of the internal Schiff's base.     

Stereochemistry and mechanism of reactions catalyzed by tryptophanase Escherichia coli.

Several beta replacement and alpha,beta elimination reactions catalyzed by tryptophanase from Escherichia coli are shown to proceed stereospecifically with retention of configuration. These conversions include synthesis of tryptophan from (2S,3R)- and (2s,3s)-[3(-3H)]serine in the presence of indole, deamination of these serines in D2O to pyruvate and ammonia, and cleavage of (2S,3R)-and (2S,3S)-[3(-3H)]tryptophan in D2O to indole, pyruvate, and ammonia. A coupled reaction with lactate dehydrogenase was used to trap the stereospecifically labeled [3-H,2H,3H]pryuvates as lactate, which was oxidized to acetate for chirality analysis of the methyl group. During deamination of tryptophan there is significant intramolecular transfer of the alpha proton of the amino acid to C-3 of indole. To determine the exposed face of the cofactor.substrate complex on the enzyme surface and to analyze its conformational orientation, sodium boro[3H]hydride was used to reduce tryptophanase-bound alaninepyridoxal phosphate Schiff's base. Degradation of the resulting pyridoxylalanine to (2S)-[2(-3H)]alanine and (4'S)-[4'(-3H)]pyridoxamine demonstrates that reduction occurs from the exposed si face at C-4' of the complex and that the ketimine double bond is trans.     

Neurospora tryptophan synthase. Characterization of the pyridoxal phosphate binding site

Tryptophan synthase, which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein that requires pyridoxal phosphate for two of its three distinct enzyme activities. Tryptophan synthase from Neurospora crassa, a homodimer of two 75-kDa subunits, was shown to bind 1 mol of pyridoxal phosphate/mol of subunit with a calculated dissociation constant for pyridoxal phosphate of 1.1 microM. The spectral properties of the holoenzyme, apoenzyme, and reconstituted holoenzyme were characterized and compared to those previously established for the heterotetrameric (alpha 2 beta 2) enzyme from Escherichia coli. The Schiff base formed between pyridoxal phosphate and the enzyme was readily reduced by sodium borohydride, but not sodium cyanoborohydride. The active site residue that binds pyridoxal phosphate, labeled by reduction of the Schiff base with tritium-labeled sodium borohydride, was determined to be lysine by high performance liquid chromatography analysis of the protein hydrolysate. A 5400-dalton peptide containing the reduced pyridoxal phosphate moiety was generated by cyanogen bromide treatment, purified and sequenced. The sequence is 85% homologous with the corresponding sequence obtained for yeast tryptophan synthase (Zalkin, H., and Yanofsky, C. (1982) J. Biol. Chem. 257, 1491-1500); the lysine derivatized by pyridoxal phosphate is located at the same relative position as that in the yeast and E. coli enzymes.     

Active-site modification of mammalian pyruvate dehydrogenase by pyridoxal 5'-phosphate

The pyruvate dehydrogenase multienzyme complex from bovine kidney and heart is inactivated by treatment with pyridoxal 5'-phosphate and sodium cyanide or sodium borohydride. The site of this inhibition is the pyruvate dehydrogenase (E1) component of the complex. Inactivation of E1 by the pyridoxal phosphate-cyanide treatment was prevented by thiamin pyrophosphate. Equilibrium binding studies showed that E1 contains two thiamin pyrophosphate binding sites per molecule (alpha 2 beta 2) and that modification of E1 increased the dissociation constant (Kd) for thiamin pyrophosphate about 5-fold. Incorporation of approximately 2.4 equiv of 14CN per mole of E1 tetramer in the presence of pyridoxal phosphate resulted in about a 90% loss of E1 activity. Radioactivity was incorporated predominantly into the E1 alpha subunit. Radioactive N6-pyridoxyllysine was identified in an acid hydrolysate of the E1-pyridoxal phosphate complex that had been reduced with NaB3H4. The data are interpreted to indicate that in the presence of sodium cyanide or sodium borohydride, pyridoxal phosphate reacts with a lysine residue at or near the thiamin pyrophosphate binding site of E1. This binding site is apparently located on the alpha subunit.     

Stereospecificity of sodium borohydride reduction of tyrosine decarboxylase from Streptococcus faecalis.

Sodium boro[3H]hydride reduction of tyrosine decarboxylase from Streptococcus faecalis followed by complete hydrolysis of the enzyme produces epsilon-[3H]pyridoxyllysine. Degradation of this material to [4'-3H]pyridoxamine and stereochemical analysis with apoaspartate aminotransferase shows that the re side at C-4' of the cofactor is exposed to solvent at pH 5.5 and 7.0. After binding of L-tyrosine at pH 5.5 or tyramine at pH 7.0 to the holoenzyme, sodium boro[3H]hydride reduction proceeds from the si face at C-4' of the substrate . cofactor complex. This indicates one of two conformational changes occurs upon binding of substrate; either rotation about the C-4 to C-4' bond in the cofactor or rotation about the axis through the C-5 and C-5' bond.     

The stereospecific removal of a C-19 hydrogen atom in oestrogen biosynthesis

1. The synthesis of a number of 19-substituted androgens is described. 2. A method for the partially stereospecific introduction of a tritium label at C-19 in 19-hydroxyandrost-5-ene-3beta,17beta-diol was developed. The 19-(3)H-labelled triol produced by reduction of 19-oxoandrost-5-ene-3beta,17beta-diol with tritiated sodium borohydride is tentatively formulated as 19-hydroxy[(19-R)-19-(3)H]androst-5-ene-3beta,17beta-diol and the 19-(3)H-labelled triol produced by reduction of 19-oxo[19-(3)H]-androst-5-ene-3beta,17beta-diol with sodium borohydride as 19-hydroxy[(19-S)-19-(3)H]-androst-5-ene-3beta,17beta-diol. 3. In the conversion of the (19-R)-19-(3)H-labelled compound into oestrogen by a microsomal preparation from human term placenta more radioactivity was liberated in formic acid (61.6%) than in water (38.4%). In a parallel experiment with the (19-S)-19-(3)H-labelled compound the order of radioactivity was reversed: formic acid (23.4%), water (76.2%). 4. These observations are interpreted in terms of the removal of the 19-S-hydrogen atom in the conversion of a 19-hydroxy androgen into a 19-oxo androgen during oestrogen biosynthesis. 5. It is suggested that the removal of C-19 in oestrogen biosynthesis occurs compulsorily at the oxidation state of a 19-aldehyde with the liberation of formic acid.     

Reductive trapping of substrate to bovine plasma amine oxidase

Plasma amine oxidases catalyze the oxidative deamination of amines to aldehydes, followed by a 2e- reduction of O2 to H2O2. Pyrroloquinoline quinone (PQQ), previously believed to be restricted to prokaryotes, has recently been proposed to be the cofactor undergoing reduction in the first half-reaction of bovine plasma amine oxidase (Ameyama, M., Hayashi, U., Matsushita, K., Shinagawa, E., and Adachi, O. (1984) Agric. Biol. Chem. 48, 561-565; Lobenstein-Verbeek, C. L., Jongejan, J. A., Frank, J., and Duine, J. A. (1984) FEBS Lett. 170, 305-309). This result is unexpected, since model studies with PQQ implicate Schiff's base formation between a reactive carbonyl and substrates, whereas experiments with bovine plasma amine oxidase have failed to provide evidence for a carbonyl cofactor. We have, therefore, re-examined putative adducts between substrate and enzyme-bound cofactor, employing a combination of [14C]benzylamine and [3H]NaCNBH3. The use of the relatively weak reductant, NaCNBH3, affords Schiff's base specificity and permits the study of enzyme below pH 7.0. As we show, enzyme can only be inactivated by NaCNBH3 in the presence of substrate, leading to the incorporation of 1 mol of [14C]benzylamine/mol of enzyme subunit at complete inactivation. By contrast, we are unable to detect any labeling with [3H]NaCNBH3, analogous to an earlier study with [3H]NaCNBH4 (Suva, R. H., and Abeles, R. H. (1978) Biochemistry 17, 3538-3545). We conclude, first, that our inability to obtain adducts containing both carbon 14 and tritium rules out the reductive trapping either of amine substrate with pyridoxal phosphate or of aldehyde product with a lysyl side chain and, second, that the observed pattern of labeling is fully consistent with the presence of PQQ at the active site of bovine plasma amine oxidase.     

The orientation and accessibility of substrates on the active site of triosephosphate isomerase.

Tritiated sodium borohydride was used to reduce the substrates of triosephosphate isomerase in the presence of the enzyme, and the mixture of the four possible products (D-[1(R)-3H]; D-[1(S)-3H]-; D-[2-3H]-, and L-[2-3H]glycerol 3-phosphate) was analyzed. While enzyme-bound dihydroxyacetone phosphate is reduced completely stereoselectively and at a rate eight imes faster than in free solution, D-glyceraldehyde 3-phosphate is inaccessible to reduction by borohydride when bound to the active site of the enzyme.     

The catalytic mechanism of tryptophan synthase from Escherichia coli. Kinetics of the reaction of indole with the enzyme--L-serine complexes

The mechanism by which indole condenses with L-serine in the active site of tryptophan synthase was studied by the stopped-flow technique. The single turnover occurs by rapid binding of indole to the pre-formed enzyme--L-serine complex, followed by C--C bond formation, reprotonation of the alpha carbon carbanion of L-tryptophan, and its final release. The effects of isotopic substitution at C-3 of indole, of pH, and of the presence of indolepropanol phosphate on these processes were also studied. The mechanism of binding of indole complements the known mechanisms of binding of L-serine and L-tryptophan to give a detailed picture of the mechanism of catalysis. It invokes two competent species of enzyme--L-serine complexes, leading to a branched pathway for the central condensation process. The rates of dehydration of L-serine and reprotonation of the carbanion of L-tryptophan are probably limited by rearrangements at the active site. Analysis of absorption, fluorescence and circular dichroic spectra, as well as of published data on the stereoisomers obtained by reduction with borohydride, suggests that the rearrangement includes a reorientation of the pyridoxal phosphate C-4' atom. The mechanism provides a detailed framework for explaining all available information, including the activating effect of the alpha subunit on the reaction catalyzed by the beta 2 subunit.     

Stereochemistry of 1,2-hydrogen loss during aromatization in the brain

The stereochemistry of hydrogen loss from C-1 and C-2 during aromatization in rat brain was studied using androstenedione containing a known distribution of isotopic label. Comparison of the tritium content of the estrone obtained from the aromatization of androstenedione labeled predominantly in the 1 alpha,2 alpha positions with that in estrone obtained from a parallel incubation using substrate with label in the 1 beta,2 beta orientation gave an estrone alpha/beta ratio of 3.6. This ratio compares with a calculated value of 4.3 for an aromatization mechanism involving loss of the 1 beta,2 beta-hydrogens. The distortion from the predicted value is due to the loss of tritium from the alpha-substrate which is unrelated to aromatization. The ratio determined experimentally is compatible with 2 beta-tritium loss since random or alpha-elimination from C-2 would yield alpha/beta ratios of 2.2 and 1.3 respectively. In an analogous manner the stereochemistry of tritium loss at C-1 was determined using [1 alpha-3H] and [1 beta-3H]androstenedione. The alpha/beta ratio of the isolated estrone was 3.6 which is in good agreement with the calculated value of 3.3 for 1 beta-tritium elimination. Our results therefore show that estrogen formation in the brain occurs with the same stereospecificity of hydrogen loss at C-1 and C-2 as in placental microsomes.     

Evidence for the extramembranous location of the putative amphipathic helix of acetylcholine receptor

Evidence has been obtained demonstrating that the peptides GVKYIAE and AIKYIAE found in the potential amphipathic helices of the alpha and beta subunits, respectively, of acetylcholine receptor are not buried in the membrane. The peptide KYIAE was synthesized, and polyclonal antibodies were prepared against a conjugate of bovine serum albumin and synthetic peptide. An immunoadsorbent capable of binding and subsequently releasing peptides ending with the sequence-YIAE was produced by attaching these specific antibodies to agarose. Native acetylcholine receptor was labeled with pyridoxal phosphate and Na[3H]BH4. The labeled protein was stripped of phospholipid and digested with the protease from Staphylococcus aureus strain V8. The digest was submitted to immunoadsorption to isolate the labeled indigenous peptides. As a control, alpha and beta polypeptides prepared by gel filtration of a solution of acetylcholine receptor in detergent were stripped of detergent and labeled with pyridoxal phosphate and Na[3H]BH4 in the presence of 8 M urea. The labeled alpha and beta polypeptides were digested and submitted to immunoadsorption. The specific radioactivities of the indigenous peptides from the alpha and beta subunits labeled under native and denaturing conditions were nearly equal. In similar experiments using isethionyl (2', 4'-dinitrophenyl)-3-amino-propionimidate as the labeling agent, the indigenous peptides from native and denatured receptor were also labeled to the same extent. Since these peptides are labeled to the same extent whether or not the protein is denatured, they cannot be buried in the membrane.     

Pyridoxamine-5'-phosphate oxidase exhibits no specificity in prochiral hydrogen abstraction from substrate

The stereochemistry for hydrogen removal from pyridoxamine 5'-phosphate with liver pyridoxine (pyridoxamine)-5'-phosphate oxidase was examined to determine whether or not there are significant steric constraints at the substrate region of the active site of the oxidase. For this, pyridoxal 5'-phosphate was reduced with tritium-labeled sodium borohydride in ammoniacal solution to yield racemically labeled [4',4'-3H]pyridoxamine 5'-phosphate which was then chemically or enzymatically oxidized to [4'-3H]pyridoxal 5'-phosphate. This latter was used as coenzyme with either L-aspartate (L-glutamate) aminotransferase and L-glutamate or L-glutamate decarboxylase and alpha-methyl-DL-glutamate to generate [4'-3H]pyridoxamine 5'-phosphate known to be labeled in the R-position. Reaction of the oxidase with the pro-R as well as the pro-R,S-labeled substrates followed by isolation of [4'-3H]pyridoxal 5'-phosphate and 3H2O revealed only half the radioactivity was abstracted from the original substrate in either case. Hence, the oxidase is not stereospecific and equally well catalyzes removal of either pro-R or pro-S hydrogen from the 4-methylene of pyridoxamine 5'-phosphate.     

Biosynthesis of riboflavin: mechanism of formation of the ribitylamino linkage

Feeding experiments with Ashbya gossypii followed by NMR analysis of the resulting riboflavin showed incorporation of deuterium from D-[2-2H]ribose at C-2' and from D-[1-2H]ribose in the pro-R position at C-1' of the ribityl side chain. The results rule out an Amadori rearrangement mechanism for the reduction of the ribosylamino to the ribitylamino linkage and point to formation of a Schiff base that is reduced stereospecifically opposite to the face from which the oxygen has departed. As prerequisite for the analysis, the 1H NMR signals for the pro-R and pro-S hydrogens at C-1' of 6,7-dimethyl-8-ribityllumazine and riboflavin and its tetraacetate were assigned with the aid of synthetic stereospecifically deuteriated samples.     

Inactivation of yeast fatty acid synthetase by modifying the beta-ketoacyl reductase active lysine residue with pyridoxal 5'-phosphate

Treatment of yeast fatty acid synthetase with pyridoxal 5'-phosphate inhibited the enzyme. Assays of the partial activities of the pyridoxal phosphate-treated synthetase showed that only the beta-ketoacyl reductase was significantly inhibited. NADPH prevented inactivation of the enzyme by pyridoxal phosphate, indicating that pyridoxal modifies a residue near or in the beta-ketoacyl reductase site. The pyridoxal-treated synthetase shows a fluorescence spectrum with a maximum of 426 nm after uv irradiation at 325 nm. Binding of the pyridoxal phosphate to the synthetase is reversible as shown by the disappearance of the fluorescence band after dialysis of pyridoxal-treated enzyme. Reduction with NaBH4 of the pyridoxal-treated enzyme eliminates this fluorescence maximum and causes the appearance of a new band at 393 nm. These observations suggest that pyridoxal phosphate interacts with the synthetase by forming a Schiff base with lysine residue at the beta-ketoacyl reductase site. Amino acid analyses of the HCl hydrolysates of the borohydride-reduced, pyridoxal-treated synthetase showed the presence of 6 mol of N6-pyridoxal derivative of lysine per mole of fatty acid synthetase, indicating the presence of six sites of beta-ketoacyl reductase in the native enzyme. Autoradiography of sodium dodecyl sulfate-polyacrylamide gels of the pyridoxal phosphate enzyme reduced with NaB3H4 indicates that the alpha subunit contains the beta-ketoacyl reductase domain. These findings are consistent with the proposed structure of the alpha 6 beta 6 complex required for palmitoyl-CoA synthesis.     

Modification of Rhodospirillum rubrum ribulose bisphosphate carboxylase with pyridoxal phosphate. 1. Identification of a lysyl residue at the active site.

Ribulose 1,5-bisphosphate carboxylase isolated from Rhodospirillum rubrum was strongly inhibited by low concentrations of pyridoxal 5'-phosphate. Activity was protected by the substrate ribulose bisphosphate and to a lesser extent by other phosphorylated compounds. Pyridoxal phosphate inhibition was enhanced in the presence of magnesium and bicarbonate, but not in the presence of either compound alone. Concomitant with inhibition of enzyme activity, pyridoxal phosphate forms a Schiff base with the enzyme which is reversible upon dialysis and reducible with sodium borohydride. Subsequent to reduction of the Schiff base with tritiated sodium borohydride, tritiated N6-pyridoxyllysine could be identified in the acid hydrolysate of the enzyme. Only small amounts of this compound were present when the reduction was performed in the presence of carboxyribitol bisphosphate, an analogue of the intermediate formed during the carboxylation reaction. Therefore, it is concluded that pyridoxal phosphate modifies a lysyl residue close to or at the active site of ribulose bisphosphate carboxylase.     

Biosynthesis of biopterin. Studies on the mechanism of 6-pyruvoyltetrahydropteridine synthase

[1'-3H]- and [2'-3H]dihydroneopterin triphosphate (NH2TP) were prepared enzymatically from [4-3H]- and [5-3H]glucose and converted to tetrahydrobiopterin (BH4) by an extract from bovine adrenal medulla. The formation of BH4 from both [1'-3H]- and [2'-3H]-NH2TP proceeds with virtually complete loss of the respective tritium label. The breaking of the CH-bond at C-1' is characterized by a kinetic isotope effect of 2.6 +/- 0.5. A smaller kinetic isotope effect of 1.5 +/- 0.2 was found for the breaking of the CH-bond at C-2'.     

Stereoselective reduction of C-2 substituted steroid C-3 ketones with lithium tris-(R,S-1,2-dimethylpropyl)-borohydride and sodium borohydride

The effect of C-2 substitution on the stereoselective reduction of steroid C-3 ketones with lithium tris-(R,S-1,2-dimethylpropyl)-borohydride and sodium borohydride was investigated. The C-2 mono- and di-substituted chloro and methyl derivatives were predominantly reduced to one of the epimeric alcohols. The 2 alpha-chloro and 2 alpha-methyl derivatives of 17 beta-acetoxy-5 alpha-androstan-3-one undergo stereoselective reduction with lithium tris-(R,S-1,2-dimethylpropyl)-borohydride to the axial (3 alpha) alcohol as observed in the unsubstituted compound, whereas sodium borohydride gives predominantly the equatorial (3 beta) alcohol. The 2 beta-chloro, 2 beta-methyl, 2,2-dichloro, and 2,2-dimethyl derivatives are reduced predominantly to the equatorial (3 beta) alcohol by both reagents.     

The isolation and characterization of 60 nm vesicles (''nanovesicles'') produced during ionophore A23187-induced budding of human erythrocytes.

1. Penicillin N was synthesized by coupling alpha-amino-alpha-p-nitrobenzyl-N-p-nitro-benzyloxycarbonyl-D-adipate with 6-aminopenicillanic acid benzyl ester, followed by removal of the protecting groups through hydrogenolysis. 2. alpha-Amino-alpha-p-nitrobenzyl-N-p-nitrobenzyloxycarbonyl-D-[5-14C]adipate was prepared by treating alpha-p-nitrobenzyl-N-p-nitrobenzyloxycarbonyl-D-glutamic acid with [14C]diazomethane followed by rearrangement with silver trifluoromethanesulphonate. 3. Coupling of alpha-amino-alpha-p-nitrobenzyl-N-p-nitrobenzyloxycarbonyl-D-[5-14C]adipate with 6-aminopenicillanic acid benzyl ester gave triprotected [10-14C]penicillin N. 4. 3H was introduced at C-6 of the Schiff's base derivative (10) by oxidation followed by reduction with NaB3H4. 5. The so-derived (6 alpha-3H)-labelled Schiff's base was hydrolysed to give 6-amino [6 alpha-3H]penicillanic acid benzyl ester p-toluenesulphonic acid salt, which after coupling as the free amine with alpha-amino-alpha-p-nitrobenzyl-N-pnitrobenzyloxycarbonyl-D-adipate and then hydrogenolysis, yielded [6alpha-3H]penicillin N. 6. Triprotected [10-14C]penicillin N and triprotected [6alpha-3H]penicillin N in admixture were hydrogenolysed to give [10-14C,6alpha-3H]penicillin N.     

Purification and properties of Escherichia coli 4'-phosphopantothenoylcysteine decarboxylase: presence of covalently bound pyruvate

4'-Phosphopantothenoylcysteine decarboxylase was purified 900-fold from Escherichia coli B with an overall yield of 6%. The enzyme migrates as a single band with a molecular weight of 35,000 +/- 3000 in 10% polyacrylamide gel electrophoresis under denaturing conditions. The native enzyme has an apparent molecular weight of 146,000 +/- 9000 as determined by a gel exclusion column. At pH 7.6 and 25 degrees C, Km = 0.9 mM and Vmax = 600 nmol/(min X mg of protein). The pH optimum for Vmax is between 7.5 and 7.7. Hydroxylamine, phenylhydrazine, potassium cyanide, and sodium borohydride as well as pyridoxal phosphate and pyridoxal inactivated the enzyme. The enzyme contains covalently bound pyruvate as suggested by the isolation of [3H]lactate and pyruvate from [3H]NaBH4-reduced enzyme and native enzyme, respectively. One mole of [3H]lactate was isolated per 39,000 g of [3H]NaBH4-reduced and completely inactivated enzyme, and 1 mol of pyruvate was isolated per 31,000 +/- 4000 g of native enzyme. Mild base treatment released lactate and pyruvate from the reduced and the native enzymes, respectively, suggesting the pyruvate is attached to the enzyme by an ester bond. These findings are in accord with similar results obtained with the horse liver enzyme (R. Scandurra, personal communication). The presence of covalently bound pyruvate in the bacterial and mammalian enzymes suggests that pyruvate plays a major role in the mechanism of action.     

The identity of a cyanogen bromide fragment of bovine dentin collagen containing the site of an intermolecular cross-link.

A peptide fraction isolated from a cyanogen bromide digest of bovine dentin collagen had a molecular weight of 46000. Its size and amino acid composition indicated that it could not consist of peptides derived from the cleavage of a single alpha chain. On reduction with tritiated sodium borohydride, radioactivity was incorporated primarily into 5, 5'-dihydroxylysinonorleucine without degradation at the peptide backbone. Periodate cleavage of the reduced or nonreduced peptide fraction generated one fragment of molecular weight 28000 and one of 18000 completely accounting for the size of the parent peptide. On amino acid analysis the constituent single-chain peptides were determined to be alpha2CB4 and alpha1CB6. Both peptides isolated after periodate oxidation of the tritiated borohydride reduced cross-link peptide were found to contain (3H)hydroxynorvaline. These data show that some hydroxylysine of alpha2CB4, a helical region peptide, was present in aldehyde form and could act as the aldehyde donor icross-link, Schiff's base formation. The only cross-linkage of this alpha2CB4 acting as an aldehyde donor peptide to alpha1CB6 would be a helical region to helical region bond, perhaps accounting for the unusual stability and low solubility of dentin collagen.