The composition and characteristics of skin and muscle collagens from a freshwater catfish grown in biologically treated tannery effuent water

The skin of catfish Gariepinus spp. reared in tannery effuent water (ETP) had an increased collagen content, decreased acid solubility and high carbohydrate association as compared with skin of normal fish. The skin collagen in either fish was composed of three distinct α chains with mobilities different from that of vertebrate type I α chains and two of these three chains were invariably present in the muscle collagen. The amino acid composition of ETP fish skin and muscle collagens were similar but were characterized by higher degrees of proline and lysine hydroxylation, indicating higher stability. Both the skin and muscle collagens had significantly high denaturation temperatures. Electron microscopy of in vitro reconstituted fibrils of skin and muscle collagens of ETP fish displayed defined periodicities of around 64 nm. This demonstrates that the polymorphism in skin collagen chains is not confined to marine fish. There was a close similarity in the composition of skin and muscle collagens and the ETP environment influences the solubility and stability of the skin collagen. It is hypothesized that the third chain may play a role in the anchorage of skin to muscle, as such chain composition is now evident in fish such as hake, cod and catfish where the musculature is strongly attached to the skin.     

Physico-chemical studies of micelle formation on sepia cartilage collagen solutions in acetate buffer and its interaction with ionic and nonionic micelles. Hydrodynamic and thermodynamic studies

Sepia cartilage collagen (pepsin-extracted) in acetate buffer (pH = 2.98) forms micelles at a particular concentration below which they do not normally form. The critical micelle concentration (cmc) of the collagen was determined in buffer as well as in SDS, cetyltrimethylammonium bromide (CTAB) and Tween-80 micellar environments at different temperatures. Mutual interaction of collagen micelles with the ionic and nonionic micelles through the formation of the mixed micelle concept has also been found. The cmc of collagen decreased in the presence of SDS and Tween-80 micelles whereas it increased in the presence of CTAB micelles. This clearly suggests that the micelle formation of collagen is facilitated by the presence of SDS and Tween-80 and hindered by CTAB micelles. The various thermodynamic parameters were estimated from viscosity measurements and the transfer of collagen into the micelles of various surfactants and the reverse phenomenon was analyzed. This analysis has also been modelled conceptually as a different phase and the results have supported the above phenomenon. Our thermodynamic results are also able to predict the exact denaturation temperature as well as the structural order of water in the collagen in various environments. The hydrated volumes, Vh, of collagen in the above environments and intrinsic viscosity were also calculated. The low intrinsic viscosity, [eta], of collagen in an SDS environment compared to buffer and other surfactant environments suggested more workable systems in cosmetic and dermatological skin care preparations. The one and two-hydrogen-bonded models of this collagen in various environments have been analyzed. The calculated thermodynamic parameters varied with the concentration of collagen. The change of thermodynamic parameters from coil-coil to random-coil conformation upon denaturation of collagen were calculated from the amount of proline and hydroxyproline residues and compared with viscometric results. Thermodynamic results suggest that the stability of the collagen in the additive environments is in the following order: SDS greater than Tween-80 greater than buffer greater than CTAB.     

Heat denaturation of human high density lipoproteins and chylomicrons

1. The objective of this investigation was to determine whether structural differences between apolipoproteins could be detected by heat denaturation. 2. The apoproteins of human serum high density lipoprotein (HDL2, d = 1.070-1.125 and HDL3, d = 1.125-1.21 g/ml), their major polypeptide constituents (R-Thr and R-Gln), and apochylomicrons were investigated. 3. Heat denaturation was found to be reversible in the temperature range from 20 to 80 degrees. 4. The thermodynamic parameters of heat denaturation delta F, delta H, delta S and delta Cp were calculated on the basis of a single transition from the "native" to "denatured" state for apo-HDL2, apochylomicrons, R-Thr and R-Gln; for apo-HDL3 these parameters were calculated on the basis of two transitions. 5. The thermodynamic parameters, with the exception of delta F, which describe heat denaturation of high density apolipoprotein, of high density apolipoprotein polypeptides and of apochylomicrons were found to be similar on a molar basis and to have approximately the same values as the thermodynamic parameters which describe heat denaturation of non-lipid binding proteins; on a weight basis differences were apparent between the apolipoproteins and the polypeptides or non-lipid binding proteins.     

A new approach to the thermodynamic role of imino acids in collagen. Solution of a thermodynamic paradox]

The dependence of denaturation transition thermodynamic parameters in various collagens from imino acid compositions has been analysed. Computational and experimental data suggest independence of the collagen molecule hydration on imino acid composition and sequence in the polypeptide chain. The continuous net of hydrogen bonds is interrupted, if imino acid residues occur in the sequence of amino acid residues, as follows from Monte Carlo computations, because the hydrogen of NH-group plays sufficient role in water shell formation for this conformation. As a consequence, entropy of denatured collagen-water system increases hand by hand with increasing imino acid content and therefore delta S increases. The increase of enthalpy of transition from imino acid content is determined by favorable Van der Waals interactions of pyrrolidine rings in native triple helical collagen structure. It was pointed out that proline role is determined by decreasing hydration in the single stranded polypeptide chain in Polyproline II conformation that leads to an increase of entropy of the polypeptide-water system. Thus, the collagen structure formation by imino acids is promoted in the water media due to single chain left-helical conformation being unfavorable for proline residues as well as due to the enthalpy nature of the triple helix stabilization.     

Physical properties of type I collagen extracted from fish scales of Pagrus major and Oreochromis niloticas

Type I collagens were extracted from fish scales of Pagrus major and Oreochromis niloticas as a possible underutilized resource for medical materials. The fish scales were demineralized with EDTA and digested by pepsin. The resultant type I collagens contained more than 33.6% of glycine as the most abundant amino acid. The denaturation temperatures of the collagens from P. major and O. niloticas were 303 and 308 K, respectively, both of which were relatively lower than that of porcine dermis collagen (314 K). CD spectra indicated that the denaturation temperatures were dependent on the amount of hydroxyproline, rather than proline residues. Raman spectra also indicated that the relative intensities of Raman lines at 879 and 855 cm⁻¹ assigned to Hyp and Pro rings were changed due to the contents of the imino acids. Significantly, the content of sulphur-containing methionine was higher in the fish scales than in porcine dermis. The enthalpy and entropy estimated from thermal analyses could be correlated to amino acid sequences (Gly-Pro-Hyp) of type I collagens and the number of methionine amino acid residues.     

鲤鱼鱼皮胶原蛋白的提取及其性质研究

首先对鲤鱼鱼皮的成分进行了研究,通过凯氏定氮、索式抽提、高温灰化、直接干燥法测定了鱼皮的蛋白质、脂肪、灰分、水分含量。通过粘度测定法得到鱼皮胶原蛋白的变性温度为28℃左右;紫外可见光谱扫描的结果证明鱼皮胶原蛋白在228 nm有一最大吸收峰;SDS-PAGE电泳初步确定鱼皮胶原蛋白是I型胶原蛋白;酶解实验进一步证实了鱼皮同鱼鳞、鱼骨的胶原蛋白在结构上具有很大的相似性。     

Temperature dependence of the preferential interactions of ribonuclease A in aqueous co-solvent systems: thermodynamic analysis.

The temperature dependence of preferential solvent interactions with ribonuclease A in aqueous solutions of 30% sorbitol, 0.6 M MgCl2, and 0.6 M MgSO4 at low pH (1.5 and 2.0) and high pH (5.5) has been investigated. This protein was stabilized by all three co-solvents, more so at low pH than high pH (expect 0.6 M MgCl2 at pH 5.5). The preferential hydration of protein in all three co-solvents was high at temperatures below 30 degrees C and decreased with a further increase in temperature (for 0.6 M MgCl2 at pH 5.5, this was not significant), indicating a greater thermodynamic instability at low temperature than at high temperature. The preferential hydration of denatured protein (low pH, high temperature) was always greater than that of native protein (high pH, high temperature). In 30% sorbitol, the interaction passed to preferential binding at 45% for native ribonuclease A and at 55 degrees C for the denatured protein. Availability of the temperature dependence of the variation with sorbitol concentration of the chemical potential of the protein, (delta mu(2)/delta m3)T,p,m2, permitted calculation of the corresponding enthalpy and entropy parameters. Combination with available data on sorbitol concentration dependence of this interaction parameter gave (approximate) values of the transfer enthalpy, delta H2,tr, and transfer entropy delta S2,tr. Transfer of ribonuclease A from water into 30% sorbitol is characterized by positive values of the transfer free energy, transfer enthalpy, transfer entropy, and transfer heat capacity. On denaturation, the transfer enthalpy becomes more positive. This increment, however, is small relative to both the enthalpy of unfolding in water and to the transfer enthalpy of the native protein from water a 30% sorbitol solution.     

Effect of hydration on the stability of the collagen-like triple-helical structure of [4(R)-hydroxyprolyl-4(R)-hydroxyprolylglycine]10

X-ray analysis has been carried out on a crystal of the collagen model peptide (Hyp(R)-Hyp(R)-Gly)10 [where Hyp(R) is 4(R)-hydroxyproline] with 1.5 A resolution. The triple-helical structure of (Hyp(R)-Hyp(R)-Gly)10 has the same helical parameters and Rich and Crick II hydrogen bond patterns as those of other collagen model peptides. However, our full-length crystal structure revealed that almost all consecutive Hyp(R) residues take the up-up pucker in contrast to putative down-up puckering propensities of other collagen model peptides. The unique feature of thermodynamic parameters associated with the conformational transition of this peptide from triple helix to single coil is that both enthalpy and entropy changes of the transition are much smaller than those of other model peptides such as (Pro-Pro-Gly)10 and (Pro-Hyp(R)-Gly)10. To corroborate the precise structural information including main- and side-chain dihedral angles and intra- and interwater bridge networks, we estimated the degrees of hydration by comparing molecular volumes observed experimentally in solution to those calculated ones from the crystal structure. The results showed that the degree of hydration of (Hyp(R)-Hyp(R)-Gly)10 is comparable to that of (Pro-Hyp(R)-Gly)10 in the triple-helical state, but the former was more highly hydrated than (Pro-Hyp(R)-Gly)10 in the single-coil state. Because hydration reduces the enthalpy due to the formation of a hydrogen bond with a water molecule and diminishes the entropy due to the restriction of water molecules surrounding a peptide molecule, we concluded that the high thermal stability of (Hyp(R)-Hyp(R)-Gly)10 is able to be described by its high hydration in the single-coil state.     

Different effects of 4-hydroxyproline and 4-fluoroproline on the stability of collagen triple helix

Differential scanning calorimetry (DSC) analyses of a series of collagen model peptides suggest that 4-hydroxyproline (Hyp) and 4-fluoroproline (fPro) have different effects on the stability of the collagen triple helices according to the sequence of amino acids and stereochemistry at the 4 positions of these imino acids. The thermodynamic parameters indicate that the enhanced stabilities are classified into two different types: the enthalpy term is primarily responsible for the enhanced stability of the triple helix of (Pro-Hyp(R)-Gly)(10), whereas the entropy term dominates the enhanced stability of (Pro-fPro(R)-Gly)(10). The difference between the molecular volumes observed in solution and intrinsic molecular volumes calculated from the crystal structure indicates the different hydration states of these peptides. (Pro-Hyp(R)-Gly)(10) is highly hydrated compared to (Pro-Pro-Gly)(10), which contributes to the larger enthalpy. In contrast, the volume of (Pro-fPro(R)-Gly)(10) shows a smaller degree of hydration than that of (Pro-Pro-Gly)(10). The entropic cost of forming the triple helix of the fPro-containing peptides is compensated by a decrease in an ordered structure of water molecules surrounding the peptide molecule, although the contribution of enthalpy originating from the hydration is reduced. These arguments about the different contribution of entropic and enthalpic terms were successfully applied to interpret the stability of the triple helix of (fPro(S)-Pro-Gly)(10) as well.     

Parallel stranded DNA with AT base pairing

The concentration and temperature dependences of the UV and CD spectra of the oligonucleotide 3'-d(ApTpApTpApTpApTpApTp)-O(CH2)6O-5'-d(pApTpApTpApTpApT pApT) (eicosamer) in aqueous solution at pH 7 in the presence of 0.5 M NaCl were studied. At less than 10(-6) M, the eicosamer was shown to form in solution a hairpin with parallel orientation of chains (parallel hairpin). From thermal denaturation profiles [A260(T)] the thermodynamic parameters, delta H degrees, delta S degrees and Tm for parallel hairpin formation were calculated to be -90 +/- 8 kJ/mol. -300 +/- 20 J.mol-1.K-1 and 40.5 degrees C, respectively. The CD spectra of the parallel double helix differed from those of B-form DNA and had characteristic features: decreasing magnitude of the positive maximum at 265 nm and a negative peak at 285 nm.     

A 13C NMR study on collagens in the solid state: hydration/dehydration-induced conformational change of collagen and detection of internal motions.

We recorded 13C NMR spectra of type I and IV collagens in the anhydrous and hydrated states, in order to confirm our previous assignment of peaks, and to analyze the mode of partial renaturation of soluble collagens by hydration, as well as rapid intramolecular motions such as ring puckering in proline or hydroxyproline residues. First, we attempted to assign all 13C NMR peaks of collagen fibrils on the basis of computer simulation by utilizing amino-acid composition and chemical shift data from both the solid state and solution. We confirmed that some previously unassigned peaks were not ascribable to a denatured portion but to the minor amino-acid residues. The 13C NMR peaks from soluble collagens were appreciably broadened and some peaks were displaced as compared with those of intact collagen fibrils. This was caused by the presence of a partial conformational disorder and/or denaturation at the time of acid-solubilization and dehydration. Those line broadening and displacements of peaks, however, were partially removed by humidification under an atmosphere of 96% R.H. over 12 h. Furthermore, we found that the 13C spin-lattice relaxation times (T1s) of both the C beta and C gamma carbons of Pro and Hyp in fibrils are substantially reduced as compared with those of some crystalline oligopeptides. It was shown that the presence of rapid ring puckering motion in these residues results in a reduction of the NT1 values, where N stands for the number of protons attached to the carbon under consideration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isinglass/collagen: denaturation and functionality

Isinglass is widely used commercially to clarify alcoholic beverages by aggregation of the yeast and other insoluble particles. It is derived from swim bladders of tropical fish by solubilisation in organic acids and consists predominantly of the protein collagen. The low content of intermolecular cross-links allows ready dissolution of swim bladder compared to bovine hide which is cross-linked by a high proportion of stable bonds and requires enzymic digestion to solubilise. Isinglass is no longer effective as a clarifying agent if thermally denatured hence the collagenous triple helical structure must be maintained. Thermal denaturation of isinglass occurs at 29 degrees C, compared to 40-41 degrees C for mammalian collagens, primarily due to the lower hydroxyproline content. The hydroxyproline is essential for the formation of H-bonded water-bridges through the hydroxyl group and the peptide chain thereby stabilising the triple helix. Based on the lower enthalpy determined by differential scanning calorimetry we have calculated that the thermally labile domain of the isinglass molecule was 41 residues compared to 66 for mammalian collagen. The fining efficiency was unaffected by pH, chelating agents, detergents and removal of surface proteins from yeast cells. Studies on the mechanism of action of isinglass have shown that higher molecular weight aggregates that increase the length of the collagen molecules (trimers, tetramers, etc.) increase efficiency and that their surface charge are important in the clarification process. By chemical modification, we have shown that blocking positively charged groups had no effect on the fining process, whilst negative charges are clearly essential and that increasing the negative charge by succinylation increases its efficacy. Solutions of bovine hide collagen were shown to be equally effective in refining beers and standard yeast preparations. The higher thermal denaturation temperature, ready availability and reproducibility of bovine collagen preparations gives it considerable advantages over isinglass.     

Stability mutants of staphylococcal nuclease: large compensating enthalpy-entropy changes for the reversible denaturation reaction

By use of intrinsic fluorescence to determine the apparent equilibrium constant Kapp as a function of temperature, the midpoint temperature Tm and apparent enthalpy change delta Happ on reversible thermal denaturation have been determined over a range of pH values for wild-type staphylococcal nuclease and six mutant forms. For wild-type nuclease at pH 7.0, a Tm of 53.3 +/- 0.2 degrees C and a delta Happ of 86.8 +/- 1.4 kcal/mol were obtained, in reasonable agreement with values determined calorimetrically, 52.8 degrees C and 96 +/- 2 kcal/mol. The heat capacity change on denaturation delta Cp was estimated at 1.8 kcal/(mol K) versus the calorimetric value of 2.2 kcal/(mol K). When values of delta Happ and delta Sapp for a series of mutant nucleases that exhibit markedly altered denaturation behavior with guanidine hydrochloride and urea were compared at the same temperature, compensating changes in enthalpy and entropy were observed that greatly reduce the overall effect of the mutations on the free energy of denaturation. In addition, a correlation was found between the estimated delta Cp for the mutant proteins and the d(delta Gapp)/dC for guanidine hydrochloride denaturation. It is proposed that both the enthalpy/entropy compensation and this correlation between two seemingly unrelated denaturation parameters are consequences of large changes in the solvation of the denatured state that result from the mutant amino acid substitutions.     

Thermodynamics of the denaturation of lysozyme in alcohol--water mixtures.

The thermal denaturation of lysozyme was studied at pH 2 in aqueous mixtures of methanol, ethanol, and 1-propanol by high sensitivity differential scanning calorimetry (DSC). The most obvious effect of alcohols was the lowering of Td, the temperature of denaturation, increasingly with higher alcohol concentration and longer alkyl chain. Both the calorimetric and van't Hoff enthalpies of denaturation initially increased and then decreased with increasing alcohol concentration, the ratio of the two enthalpies being nearly unity, 1.007 +/- 0.011, indicating the validity of the two-state approximation for the unfolding of lysozyme in these solvent systems. The reversibility of the denaturation was demonstrated by the reversibility of the DSC curves and the complete recovery of enzymic activity on cooling. The changes in heat capacity on unfolding decreased with increasing alcohol concentration for each alcohol. Experimentally determined values of denaturation temperature and of entropy and heat capacity changes were used to derive the additional thermodynamic parameters delta G degrees and delta S degrees for denaturation as a function of temperature for each alcohol--water mixture. Comparison of the thermodynamic parameters with those reported [Pfeil, W., & Privalov, P.L. (1976) Biophys. Chem. 4, 23--50] in aqueous solution at various values of pH and guanidine hydrochloride concentration showed that these latter changes have no effect on the heat capacity changes, whereas the addition of alcohols causes a sharp decrease.     

Molecular species of collagen in the intramuscular connective tissues of the marine crab, Scylla serrata

Type V like collagens are widely distributed in marine invertebrates, particularly crustaceans and molluscs. We have been investigating the nature of collagens in the muscular tissues of crustaceans. The presence of type V like homotrimeric collagen in prawn muscle was noted before. We report here a comparative analysis of collagens purified from the pepsin digest of abdominal and pereiopod muscle tissues of the crab, Scylla serrata. The major collagen in either muscle precipitated at 1.2 M NaCl at acid pH, suggestive of a type V like property. The homotrimeric collagen was then purified to near homogeneity by precipitation with 20% ammonium sulphate. Solubility characteristics and biochemical studies indicated the leg muscle collagens to be highly crosslinked and stabilised by more bound carbohydrates, as compared to the abdominal muscle collagen. Analysis of amino acid composition revealed a close similarity to known type V collagens and the leg muscle collagen was characterised by more lysine hydroxylation and slightly reduced glycine content. The leg muscle collagen had a higher denaturation temperature and intrinsic viscosity than the abdominal muscle collagen. Our results confirm the similarity of major crustacean muscle collagens to vertebrate type V collagen. Further, the relative complexity of leg muscle collagen, unlike the abdominal muscle collagen, correlates to the specific functional requirements, where the former is involved in locomotion and preying and the latter in normal growth and development.     

Effects of hydrated water on protein unfolding

The conformational stability of a protein in aqueous solution is described in terms of the thermodynamic properties such as unfolding Gibbs free energy, which is the difference in the free energy (Gibbs function) between the native and random conformations in solution. The properties are composed of two contributions, one from enthalpy due to intramolecular interactions among constituent atoms and chain entropy of the backbone and side chains, and the other from the hydrated water around a protein molecule. The hydration free energy and enthalpy at a given temperature for a protein of known three-dimensional structure can be calculated from the accessible surface areas of constituent atoms according to a method developed recently. Since the hydration free energy and enthalpy for random conformations are computed from those for an extended conformation, the thermodynamic properties of unfolding are evaluated quantitatively. The evaluated hydration properties for proteins of known transition temperature (Tm) and unfolding enthalpy (delta Hm) show an approximately linear dependence on the number of constituent heavy atoms. Since the unfolding free energy is zero at Tm, the enthalpy originating from interatomic interactions of a polypeptide chain and the chain entropy are evaluated from an experimental value of delta Hm and computed properties due to the hydrated water around the molecule at Tm. The chain enthalpy and entropy thus estimated are largely compensated by the hydration enthalpy and entropy, respectively, making the unfolding free energy and enthalpy relatively small. The computed temperature dependences of the unfolding free energy and enthalpy for RNase A, T4 lysozyme, and myoglobin showed a good agreement with the experimental ones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature dependence of the dielectric properties of blood

The aim of the work was to investigate the temperature effect in physiological range on the dielectric properties of a fish (Cyprinus carpio) blood. By applying an empirical non-damaging method and statistical analysis of the dielectric data, it is observed that a heating of carp blood induces an almost linear value variation of the electrical conductivity and permittivity of the intracellular matter of carp red blood cells. The dielectric data, however, reveal anomalous behaviour for conductivity and permittivity of the interior of carp erythrocytes at 23 degrees C and 23 degrees, 37 degrees C, respectively and for both volume fraction and dielectric loss factor tg delta at 30 degrees, 35 degrees, 37 degrees C. For the presented dielectric parameters the temperature coefficient and the Arrhenius activation energy were estimated. Moreover, this work led to conclusion that the changes of the volume fraction of carp erythrocytes induce corresponding variations of the maximum of the dielectric loss factor tg delta determined for a whole blood.     

The increase in denaturation temperature following cross-linking of collagen is caused by dehydration of the fibres

Differential scanning calorimetry (DSC) was used to study the thermal stability of native and synthetically cross-linked rat-tail tendon at different levels of hydration, and the results compared with native rat-tail tendon. Three cross-linking agents of different length between functional groups were used: malondialdehyde (MDA), glutaraldehyde and hexamethylene diisocyanate (HMDC). Each yielded the same linear relation between the reciprocal of the denaturation temperature in Kelvin, T(max), and the water volume fraction, epsilon (1/T(max)=0.000731epsilon+0.002451) up to a critical hydration level, the volume fraction of water in the fully hydrated fibre. Thereafter, water was in excess, T(max) was constant and the fibre remained unchanged, no matter how much excess water was added. This T(max) value and the corresponding intrafibrillar volume fraction of water were as follows: 84.1 degrees C and 0.48 for glutaraldehyde treated fibres, 74.1 degrees C and 0.59 for HMDC treated fibres, 69.3 degrees C and 0.64 for MDA treated fibres, and 65.1 degrees C and 0.69 for untreated native fibres. Borohydride reduction of the native enzymic aldimines did not increase the denaturation temperature of the fibres. As all samples yielded the same temperature at the same hydration, the temperature could not be affected by the nature of the cross-link other than through its effect on hydration. Cross-linking therefore caused dehydration of the fibres by drawing the collagen molecules closer together and it was the reduced hydration that caused the increased temperature stability. The cross-linking studied here only reduced the quantity of water between the molecules and did not affect the water in intimate contact with, or bound to, the molecule itself. The enthalpy of denaturation was therefore unaffected by cross-linking. Thus, the "polymer-in-a-box" mechanism of stabilization, previously proposed to explain the effect of dehydration on the thermal properties of native tendon, explained the new data also. In this mechanism, the configurational entropy of the unfolding molecule is reduced by its confinement in the fibre lattice, which shrinks on cross-linking.     

Enthalpic denaturation of collagen satisfies the general relation of the change in Delta H(d) from Delta C(p) for proteins at zero hydroxyproline level]

One of the noticeable peculiarities of thermodynamics of collagen is an anomalous high magnitude of the enthalpy of denaturation delta Hd at a very low thermostability. Taking into account recent ideas about the role of hydrophobic interactions in determination of the thermodynamic function of protein denaturation, it is shown that the high magnitude of delta Hd of collagen in comparison with those of globular proteins can be explained by two factors: a significant contribution of residues of 4-hydroxyproline and small magnitude of hydrophobic interactions.     

Complete primary structure of rainbow trout type I collagen consisting of alpha1(I)alpha2(I)alpha3(I) heterotrimers.

The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be alpha1(I)alpha2(I)alpha3(I) and [alpha1(I)](2)alpha2(I), respectively. The occurrence of alpha3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 degrees C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of proalpha(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each proalpha(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific alpha(I) chain, proalpha3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for proalpha1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in proalpha3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proalpha3(I) had diverged from proalpha1(I). This study is the first report of the complete primary structure of fish type I procollagen.