[Development and evaluation of a chemiluminescence immunoassay for type â procollagen N-terminal peptide].

To develop a magnetic nanoparticle chemiluminescence immunoassay (CLIA) for the determination of type Ⅰ procollagen N-terminal peptide (PINP) in human serum, we expressed a recombinant PINP-α1 protein in Corynebacterium glutamicum and used it as an immunogen to immunize BALB/c mice. We obtained three hybridoma cell lines that stably secret antibody against PINP-α1 protein. After further pairing and screening, we chose a monoclonal antibody 8C12 coupled with biotin as the capture antibody, and a monoclonal antibody 1F11 labeled horseradish peroxidase as the detection antibody. The antibodies combined with the serum samples, forming a sandwich complex which was used to detect the concentration of PINP in serum. After optimizing the conditions, we determined that the best working concentration of the capture antibody and the detection antibody were 3 µg/mL, and the incubation time was 30 minutes. The quantitative assay had a detection range of 5-1 100 ng/mL, with recovery rates between 93%-107% and the minimum detection limit of 1.22 ng/mL achieved. The intra-and inter-assay precisions were lower than 10%. The correlation coefficient of PINP results between this CLIA method and the Roche electrochemiluminescence immunoassay system was 0.906 2. Therefore, this CLIA method is specific and can be used to quantitatively detect the content of PINP in serum, which has the potential to become an auxiliary approach for bone disease examination.

Development and evaluation of a chemiluminescence immunoassay for type Ⅰ procollagen N-terminal peptide / 生物工程学报

To develop a magnetic nanoparticle chemiluminescence immunoassay (CLIA) for the determination of type Ⅰ procollagen N-terminal peptide (PINP) in human serum, we expressed a recombinant PINP-α1 protein in Corynebacterium glutamicum and used it as an immunogen to immunize BALB/c mice. We obtained three hybridoma cell lines that stably secret antibody against PINP-α1 protein. After further pairing and screening, we chose a monoclonal antibody 8C12 coupled with biotin as the capture antibody, and a monoclonal antibody 1F11 labeled horseradish peroxidase as the detection antibody. The antibodies combined with the serum samples, forming a sandwich complex which was used to detect the concentration of PINP in serum. After optimizing the conditions, we determined that the best working concentration of the capture antibody and the detection antibody were 3 μg/mL, and the incubation time was 30 minutes. The quantitative assay had a detection range of 5-1 100 ng/mL, with recovery rates between 93%-107% and the minimum detection limit of 1.22 ng/mL achieved. The intra-and inter-assay precisions were lower than 10%. The correlation coefficient of PINP results between this CLIA method and the Roche electrochemiluminescence immunoassay system was 0.906 2. Therefore, this CLIA method is specific and can be used to quantitatively detect the content of PINP in serum, which has the potential to become an auxiliary approach for bone disease examination.

Measurement of aminoterminal propeptide of type I procollagen (PINP) in serum.

The aminoterminal propeptide of type I procollagen (PINP) in serum is a sensitive indicator of the synthesis of type I collagen. Four assays are available for PINP, two of them (intact PINP assays) measure the intact propeptide and the other two (total PINP assays) also detect a smaller antigen in serum. In many clinical situations, these assays give similar information, but renal insufficiency increases the concentration of the smaller antigen, influencing both the apparent concentration of PINP and assay calibration. Serum PINP is mostly affected by changes in bone metabolism. In infants and children, the concentration is much higher than in adults. Serum PINP (s-PINP) is a useful indicator of disease activity in Paget's disease of bone, in bone metastases of osteoblastic nature, and in the follow-up of treatment of osteoporosis. The IFCC and IOF recently recommended the use of s-PINP as a reference marker for bone formation in studies concerning fracture risk assessment and treatment response.

Effect of adjunctive systemic azithromycin with periodontal surgery in the treatment of chronic periodontitis in smokers: a pilot study.

BACKGROUND: Along with conventional surgical therapy, systemic antibiotics may provide more effective treatment in smokers by targeting tissue-invasive bacteria. The aim of this randomized, placebo-controlled, double-masked clinical trial was to evaluate the adjunctive effects of systemic azithromycin (AZM) in combination with periodontal pocket reduction surgery in the treatment of chronic periodontitis in smokers. METHODS: Thirty patients with a greater than one pack/day smoking habit and generalized moderate to severe chronic periodontitis were randomized to the test (surgery plus 3 days of AZM, 500 mg) or control group (surgery plus 3 days of placebo). Full-mouth probing depth (PD), clinical attachment level (CAL), bleeding on probing (BOP), gingival index (GI), plaque index, and wound healing indices (WHI) were assessed at baseline and at 2 weeks and 1, 3, and 6 months following surgical intervention. Plaque and gingival crevicular fluid were collected for trypsin-like enzyme activity (benzoyl-dl-arginine naphthylamine) and bone biomarker (crosslinked telopeptide of type I collagen [ICTP]) analyses, respectively, at baseline, 2 weeks, and 1, 3, and 6 months. RESULTS: Surgical treatment of moderate (PD = 4 to 6 mm) and deep (PD > 6 mm) pockets significantly improved clinical parameters of treated and untreated teeth (CAL gain, PD reduction, and reduction of BOP). The additional use of AZM did not enhance this improvement nor did it promote reduction of ICTP levels. Compared to the control group, the test group had significantly better WHI scores at 1 month, significantly less GI at 2 weeks, and sustained reductions of red-complex bacteria with trypsin-like enzyme activity at 3 months. For non-surgery teeth, only the test group showed significant gains in overall CAL compared to baseline. CONCLUSIONS: The findings of this pilot study demonstrated that in heavy smokers, adjunctive systemic AZM in combination with pocket reduction surgery did not significantly enhance PD reduction or CAL gain. However, the clinical value of adjunctive AZM may be appreciated by more rapid wound healing, less short-term gingival inflammation, and sustained reductions of periopathogenic bacteria. More expanded studies are recommended to better determine the clinical effects of adjunctive AZM in patients who smoke.

[Osteogenesis imperfecta as an interdisciplinary medical problem]. / Wrodzona lamliwosc kosci jako interdyscyplinarny problem medyczny.

In years 2001-2003 six children (3 boys ond 3 girls) from 2.1 to 18.7 years old with osteogenesis imperfecta (OI) were treated in our clinic. Besides clinical and radiological evaluation, densitometry and biochemical analysis of collagen were performed. Biochemical analysis was performed on cultured in vitro fibroblasts from the skin biopsies. Based on this findings 1 child was graded as type I of OI, 4 children as type III and 1 as type IV. Recognition of collagen defect helps with diagnosis and makes the decision for pharmacological treatment easier. In 1 child with the dramatic type III OI therapy with pamidronian was implemented with good result. Performed surgical treatment, intramedullar stabilisation with Rush rod, proved to be useful choice in correcting axial deformations of the lower extremities and preventing from the future fractures.

Increased postoperative concentrations of tenascin-C in wound fluid.

BACKGROUND: Tenascin-C (Tn-C) is the most studied member of a family comprising large oligomeric glycoproteins in the extracellular matrix. The function of Tn-C still is unclear, and the levels of Tn-C in human wound fluid have not been studied. METHODS: The participants in this study were 24 patients referred for elective major gastrointestinal surgery. Concentrations of Tn-C and procollagen propeptides type 1 and type 3 in serum and wound fluid were measured after surgery. RESULTS: In wound fluid, Tn-C was present on postoperative day 1, and the concentration increased from day 5 up to day 7. CONCLUSIONS: The concentration of Tn-C increases postoperatively in wound fluid. The concentration of Tn-C in wound fluid is markedly higher than that of serum. The differences in expression between Tn-C and the procollagen propeptides may reflect different tasks of these extracellular matrix proteins.

Aminoterminal propeptide of the alpha1-homotrimer variant of human type I procollagen (hotPINP) in malignant pleural effusion.

The aminoterminal propeptide (hotPINP) of type I homotrimer, a putative malignancy-associated type I collagen variant, was purified for the first time and a method was established for its detection in pleural fluid. Samples of 58 patients, with malignant or benign disease, were studied with specific immunoassays for the two propeptides of type-I procollagen (PICP and PINP) and with HPLC-DEAE chromatography to separate the two PINP variants. HotPINP was present in 64% of both benign and malignant pleural effusion fluids, with the exception of malignant mesotheliomas, none of which showed the presence of hotPINP. Also the PICP to PINP ratios were lower than normal in both benign and malignant samples (altogether in 69% of samples), although this deviation was greater in malignancy. These two phenomena were independent of each other. As synthesis of the alpha1-homotrimer-variant of type-I collagen seems to be relatively common during the formation of pleural effusion, it may be generally related to a fibroproliferative reaction in the pleural wall.

Biophysical characterization of the C-propeptide trimer from human procollagen III reveals a tri-lobed structure.

Procollagen C-propeptide domains direct chain association during intracellular assembly of procollagen molecules. In addition, they control collagen solubility during extracellular proteolytic processing and fibril formation and interact with cell surface receptors and extracellular matrix components involved in feedback inhibition, mineralization, cell growth arrest, and chemotaxis. At present, three-dimensional structural information for the C-propeptides, which would help to understand the underlying molecular mechanisms, is lacking. Here we have carried out a biophysical study of the recombinant C-propeptide trimer from human procollagen III using laser light scattering, analytical ultracentrifugation, and small angle x-ray scattering. The results show that the trimer is an elongated molecule, which by modeling of the x-ray scattering data appears to be cruciform in shape with three large lobes and one minor lobe. We speculate that each of the major lobes corresponds to one of the three component polypeptide chains, which come together in a junction region to connect to the rest of the procollagen molecule.

Keratinocyte growth factor and hepatocyte growth factor in bronchoalveolar lavage fluid in acute respiratory distress syndrome patients.

OBJECTIVES: To determine bronchoalveolar lavage (BAL) fluid concentrations of keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF), two potent growth factors for alveolar type II epithelial cells, in patients with acute respiratory distress syndrome (ARDS). DESIGN: Prospective study. SETTING: An adult trauma/surgical intensive care unit in an urban teaching hospital. PATIENTS: A total of 32 ventilated patients with pulmonary infiltrates prospectively identified with ARDS (n = 17) or without ARDS (n = 15), including eight patients with hydrostatic edema (HE), and ten nonventilated patients serving as controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: BAL was performed 2.88 days +/- 2.4, 3.5 days +/- 2.4, and 2.3 days +/- 2.2 after the lung insult in ARDS, HE, and other non-ARDS patients respectively (p = .32). KGF was detected in BAL fluid in 13 of the 17 ARDS patients (median, 31.6 pg/mL), in one patient with HE, and in none of other non-ARDS patients. In ARDS patients, detection of KGF in BAL was associated in BAL fluid with the detection of type III procollagen peptide (PIIIP), a biological marker of fibroproliferation. In ARDS patients, detection of KGF in BAL was associated with death (p = .02). HGF was detected in 15 ARDS patients (median, 855 pg/mL), in seven patients with HE (median, 294 pg/mL; p = .05 for the comparison with ARDS group), in six of other non-ARDS patients (median, 849 pg/mL; p = .32 with ARDS group). HGF concentrations were higher in nonsurvivors than in survivors (p = .01). None of the ten BAL of controls contained either KGF or HGF. CONCLUSION: KGF was detected almost exclusively in BAL fluid from ARDS patients and correlated with a poor prognosis in this group. In contrast, HGF was detected in the BAL fluid from a majority of patients with or without ARDS. Elevated HGF concentrations were associated with a poor prognosis in the overall group.

Non-disulfided pro alpha 1(IV) chain in B16 melanoma cell culture.

We have previously reported that two species of pro alpha 1(IV) collagen chains, disulfided (500 kDa) and non-disulfided (180 kDa), were produced by B16 melanoma cells (J Biochem (1994) 116; 1039-1043). The mechanism by which the non-disulfided pro alpha 1(IV) chain is produced was studied. No significant difference in prolyl hydroxylation in both polypeptides was observed. When the culture was treated with alpha, alpha 'dipyridyl', a potent inhibitor for hydroxylation of collagen, the secretion of the disulfided alpha 1(IV) chain was inhibited, but non-disulfided alpha 1(IV) chain secretion was not affected. Short pulse and pulse-chase experiments demonstrated that both chains appeared at the same time in the culture medium and the relative amounts of both chains in the medium were unaltered with increasing chase periods. These results indicate that the non-disulfided alpha 1(IV) chain is fully hydroxylated and that it's secretion is independent of it's hydroxylation level. A marked susceptibility of the 180 kDa alpha 1(IV) chain to pepsin at 4 degrees C suggests that the non-disulfided chain may be present in a denatured form.

Characterization of human type III collagen expressed in a baculovirus system. Production of a protein with a stable triple helix requires coexpression with the two types of recombinant prolyl 4-hydroxylase subunit.

An efficient expression system for recombinant collagens would have numerous scientific and practical applications. Nevertheless, most recombinant systems are not suitable for this purpose, as they do not have sufficient amounts of prolyl 4-hydroxylase activity. Pro-alpha 1 chains of human type III collagen expressed in insect cells by a baculovirus vector are reported here to contain significant amounts of 4-hydroxyproline and to form triple-helical molecules, although the Tm of the triple helices was only about 32-34 degrees C. Coexpression of the pro-alpha1(III) chains with the alpha and beta subunits of human prolyl 4-hydroxylase increased the Tm to about 40 degrees C, provided that ascorbate was added to the culture medium. The level of expression of type III procollagen was also increased in the presence of the recombinant prolyl 4-hydroxylase, and the pro-alpha 1(III) chains and alpha1(III) chains were found to be present in disulfide-bonded molecules. Most of the triple-helical collagen produced was retained within the insect cells and could be extracted from the cell pellet. The highest expression levels were obtained in High Five cells, which produced up to about 80 microg of cellular type III collagen (120 microg of procollagen) per 5 X 10(6) cells in monolayer culture and up to 40 mg/liter of cellular type III collagen (60 mg/liter procollagen) in suspension. The 4-hydroxyproline content and Tm of the purified recombinant type III collagen were very similar to those of the nonrecombinant protein, but the hydroxylysine content was slightly lower, being about 3 residues/1000 in the former and 5/1000 in the latter.

Cleavage of type I procollagen by C- and N-proteinases is more rapid if the substrate is aggregated with dextran sulfate or polyethylene glycol.

The enzymes procollagen C- and N-proteinases specifically cleave carboxyl- and amino-terminal propeptides of procollagens. After cleavage of the propeptides, the resulting collagens self-assemble into fibrils. In most previous experiments with the enzymes, the substrate was monomeric type I procollagen. Here we have prepared aggregates of type I procollagen from chick embryo tendons by using 1 to 100 micrograms/ml of 500-kDa dextran sulfate or 3 to 5% (w/v) polyethylene glycol (M(r) 3350). Aggregation of the substrate with dextran sulfate increased its rate of cleavage by purified or crude C-proteinase from chick embryo tendons 10- to 15-fold. Aggregation of the substrate with 25 to 100 microgram/ml of dextran sulfate increased the rate of cleavage by purified N-proteinase about 4-fold. The rate of cleavage by crude N-proteinase was enhanced only about 2-fold, apparently because of partial precipitation of the enzyme by dextran sulfate. Using polyethylene glycol to aggregate the substrate increased the rate of cleavage by procollagen C-proteinases 5- to 20-fold. Aggregation with polyethylene glycol also increased the rate of cleavage by purified procollagen N-proteinases 2- to 5-fold. With crude N-proteinase, the rate of cleavage was increased only 1.5-fold. The results suggest that the rate of cleavage of the substrate by both enzymes is increased by the aggregation of the substrate itself by dextran sulfate or polyethylene glycol. The increased rates of cleavage seen after aggregation of substrate can be used to develop more sensitive assays for the enzymic activities.

Ascorbate modulation of chondrocyte gene expression is independent of its role in collagen secretion.

During development and fracture repair, endochondral bone formation is preceded by an orderly process of chondrocyte hypertrophy and cartilage matrix calcification. Analysis of calcifying versus noncalcifying cartilage has identified several differences in matrix proteins; among these are appearance of a novel collagen, type X, and decreased synthesis of type II collagen, the major component of cartilage matrix. In addition, there is a marked increase in alkaline phosphatase, an enzyme expressed at high levels in all mineralizing tissues. Cultured chondrocytes can be induced to undergo these changes in gene expression and to produce calcified matrix by exposure to ascorbic acid. The mechanism by which ascorbate produces these changes has been examined by analyzing the effect of the vitamin on prehypertrophic chick embryo sternal chondrocytes. Nuclear run-on assays demonstrated that ascorbate alters mRNA levels in chondrocytes by changing the transcription rates. The fact that marked changes in mRNA levels require 1-2 days of ascorbate exposure suggested that the effect of this vitamin on gene transcription may be secondary to other, earlier ascorbate-induced effects. Since cells cultured with ascorbate produce a collagen-enriched matrix, we examined the hypothesis that transcriptional changes were secondary to altered cell-matrix interactions. Chondrocytes were cultured after attachment to tissue culture plastic, in suspension, or on plates coated with collagen type I. Comparison of alkaline phosphatase activity with and without ascorbate addition demonstrated that under all of these conditions, induction of enzyme was dependent on the presence of ascorbate. When plates containing ascorbate-conditioned chondrocyte matrix were used as substrate for naive chondrocytes, the cells continued to require ascorbate for induction of high levels of alkaline phosphatase and type X collagen mRNA. Addition of the hydroxylation inhibitor, 3,4-dehydroproline, caused marked inhibition of collagen secretion as well as accumulation of underhydroxylated collagens within the cells. However, even in the presence of this inhibitor ascorbate was effective in inducing elevated alkaline phosphatase and type X collagen. These results indicate that the ability of ascorbate to induce chondrocyte hypertrophy does not depend on production of a collagen-rich matrix.

Identification of alternatively spliced variants of type II procollagen in vitreous.

Adult and foetal bovine vitreous were analyzed for the presence of unprocessed and partially processed forms of type II procollagen. Type II procollagen can exist in two alternatively spliced forms: a short form which lacks a large part of the N-propeptide due to the splicing out of exon 2 and a long form in which exon 2 is expressed and which has a full-sized N-propeptide. Both splice variants were demonstrated and analyses of type II pN-collagen demonstrated that the long form predominated, the ratio of the long form to the short form being 5:1 in foetal vitreous and 1.5:1 in adult vitreous. In foetal vitreous 35% of the type II collagen extracted was in the pN form, but there was very little unprocessed type II procollagen or type II pC-collagen. In contrast, adult vitreous extracts contained relatively less type II pN-collagen, but contained larger amounts of type II pC-collagen and unprocessed type II procollagen.

Purification of human procollagen type I carboxyl-terminal propeptide cleaved as in vivo from procollagen and used to calibrate a radioimmunoassay of the propeptide.

We purified human procollagen type I carboxyl-terminal propeptide (PICP) that had been cleaved as in vivo from procollagen. PICP in serum-free medium from cultured human fetal fibroblasts was purified by thiophilic adsorption chromatography, low-pressure gel filtration, and HPLC gel filtration. The purity and homogeneity of the protein was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal amino acid sequencing showed that the sequences of the alpha 1 and alpha 2 chains of this PICP were identical to those of the PICP produced in vivo. The monocomponent PICP thus purified was used as calibrator in a simple equilibrium-type RIA of PICP with polyclonal antibodies raised in rabbits. The measuring range is 0.15-3.75 nmol/L, and the assay detection limit is 0.03 nmol/L. The within-run and total CVs are 2% and 4%, respectively. The reference interval for the plasma concentration of PICP in healthy women of ages > 30 years is 0.36-1.44 nmol/L (geometric mean 0.72 nmol/L, n = 154).

Synthesis of recombinant human procollagen II in a stably transfected tumour cell line (HT1080).

Apparently because the biosynthetic pathways involve eight or more highly specific post-translational enzymes, it has been difficult to obtain expression of genes for fibrillar collagens in recombinant systems. Here two constructs of the human gene for procollagen II (COL2A1) were prepared, one with about 0.5 kb of a promoter for a procollagen I gene (COL1A1) and the other with about 4 kb of the promoter for the procollagen II gene. The constructs, together with a neomycin-resistant gene, were transfected into a human tumour cell line (HT1080) that synthesizes the collagen IV found in basement membranes, but does not synthesize any fibrillar collagen. About two per 100 clones resistant to the neomycin analogue G418 synthesized and secreted human procollagen II. Milligram quantities of the recombinant procollagen II were readily isolated from the cultured medium. The recombinant procollagen II had the expected amino acid sequence as defined by nucleotide sequencing of mRNA-derived cDNA and the expected amino acid composition as defined by analysis of procollagen II that was converted into collagen II by digestion with procollagen N- and C-proteinases. Also, analysis of the carbohydrate content indicated that there was glycosylation of some of the hydroxylysine residues but no evidence of post-translational overmodification of the residues. In addition, the protein was shown to have a native conformation as assayed by a series of protease digestions. No essential differences were found between clones transfected with the COL2A1 gene construct containing the COL1A1 promoter and the similar construct containing the COL2A1 promoter in terms of number of clones synthesizing recombinant procollagen II and the levels of expression. With both constructs, the expression of the COL2A1 gene was closely related to copy number. The results demonstrated therefore that it is not essential to use a promoter for a gene normally expressed in a host cell in order to obtain gene copy-number-dependent expression of an exogenous collagen gene in stably transfected cells.

Characteristics of a 28-kDa collagenous protein extracted with guanidine from EDTA-demineralized rabbit alveolar bone.

Bone proteins in alveolar bone of mandibles from young adult rabbits (3-month-old) were extracted with 4.0 M guanidine hydrochloride (GuHCl), followed by 0.5 M ethylenediaminetetraacetate, and again with 4.0 M GuHCl (G2-ext). The proteins in the G2-ext were fractionated on a gel-filtration column, followed by an anion-exchange column in the presence of 7.0 M urea. A 28-kDa protein was isolated from the G2-ext. The purified 28-kDa protein showed intense staining with silver on SDS-PAGE slab-gel under reducing conditions. This protein was digested with bacterial collagenase, and a 19-kDa fragment appeared on the gel. However, the protein was not susceptible to reduction with cyanogen bromide. The protein did not bind to hydroxyapatite crystals in the presence of 7.0 M urea, and also did not bind to some lectins. On SDS-PAGE under non-reducing conditions, the protein migrated as two bands; a new band appeared at approximately the 85-kDa region in addition to the original 28-kDa band. The amino acid compositions of the protein were similar to those of the alpha 1-pN-propeptide of type I procollagen obtained from other tissues.

Heterogeneity of type III procollagen N-terminal peptides in BAL fluid from normal and fibrotic lungs.

Levels of the N-terminal propeptide of type III collagen (PIIINP) in bronchoalveolar lavage fluid (BALF) are thought to reflect type III collagen production by the lungs, and increased levels have been reported in patients with pulmonary fibrosis. We wanted to know more about the relative proportions of these peptides in normal BALF, whether they altered in pulmonary fibrosis, and whether lymphoid tissue is capable of clearing PIIINPs. In this study, we used a radioimmunoassay which detects the different forms of PIIINP-related antigens with equal specificity, to measure PIIINPs in serum and BALF of patients with cryptogenic fibrosing alveolitis (CFA). To investigate why PIIINP profiles in BALF differed from serum, the absolute concentration and relative proportion of PIIINPs in lymph afferent and efferent to the popliteal lymph node of a sheep were also determined. PIIINP concentrations were significantly greater in serum and BALF of patients with CFA, compared with controls. Gel chromatography indicated that serum antigen distribution, both of patients and controls, contained approximately 20% Col 1-3; the remainder being Col 1. In contrast, BALF contained Col 1-3 and Col 1, together with an antigen of high molecular weight (> 150 kD). The relative proportion of each antigen varied quite widely, but there were no apparent differences between patients and controls. The concentration of PIIINPs in afferent lymph was 295 ng.ml-1 and in efferent lymph was 104 ng.ml-1. Gel chromatography demonstrated that a significant amount of Col 1-3, together with a high molecular weight peptide, had been cleared during passage through the node.(ABSTRACT TRUNCATED AT 250 WORDS)

Deletion of a large domain in recombinant human procollagen II does not alter the thermal stability of the triple helix.

A construct of the human gene for procollagen II (COL2A1) was prepared with an internal deletion of 5 kilobases that removed 12 exons coding for 291 amino acids from near the NH2 terminus of the triple helix. The construct was then used to transfect stably a human tumor cell line (HT-1080), and clones secreting internally deleted pro alpha 1(II) chain of procollagen II were isolated. The protein was purified, and the thermal stability of the triple-helical domain was assayed by brief protease digestion. The thermal stability of the internally deleted protein was the same as that of intact collagen II even though the triple helix was 39% shorter. Additionally, the thermal stability of the collagenase A fragment was the same as that of the collagenase A fragment of normal collagen II even though it was 38% shorter. Analysis of the results suggested that the thermal stabilities of large fragments of collagen II depended primarily on their contents of -Gly-Pro-Hyp-triplets corrected for length.