title: Kinetic Analysis of the Digestion of Bovine Type I Collagen Telopeptides with Porcine Pepsin.
abstract: Collagen is frequently digested using pepsin in industries to produce a triple helical collagen without the N- and C-terminal telopeptides. However, kinetic analysis of this reaction is difficult because several Lys residues in the N- and in the C-terminal telopeptides form covalent bonds, leading to multiple substrates species, and pepsin cleaves collagen at various sites in the N-terminal and in the C-terminal telopeptides, yielding different products. Here we performed kinetic analysis of the digestion of bovine type I collagen with porcine pepsin. The reaction could be monitored by SDS-PAGE by measuring the intensity of the protein bands corresponding to the variant β11 chain. We obtained kinetic parameters relative to the decrease in the variant β11 chain upon digestion. At pH 4.0, the Km and kcat values increased with increasing temperature (30 to 65 °C), although the kcat /Km values were stable. Additional cleavage at the helical region was detected at 45 to 65 °C. At 37 °C, the Km and kcat values increased with decreasing pH, and the kcat /Km values at pH 2.1 to 4.5 were stable and higher than those at pH 5.0 and 5.5. No additional cleavage was detected at the examined pH. Thus, the optimal pH and temperatures for selective digestion of collagen telopeptides with pepsin are 2.1 to 4.5 and 30 to 40 °C, respectively. These results suggest that the method might be useful for the kinetic analysis of the digestion of collagen telopeptides with pepsin.
authors.affiliation: Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are with Div. of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto Univ, Sakyo-ku, Kyoto 606-8502, Japan.|Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are also with Nippi Research Inst. of Biomatrix, 520-11, Kuwabara, Toride, Ibaraki, 302-0017, Japan.|Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are also with Medical Research Support Center, Graduate School of Medicine, Kyoto Univ, Yoshida Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Qian
authors.affiliation: Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are with Div. of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto Univ, Sakyo-ku, Kyoto 606-8502, Japan.|Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are also with Nippi Research Inst. of Biomatrix, 520-11, Kuwabara, Toride, Ibaraki, 302-0017, Japan.|Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are also with Medical Research Support Center, Graduate School of Medicine, Kyoto Univ, Yoshida Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: Yukari
authors.initials: Y
authors.lastname: Okada
authors.affiliation: Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are with Div. of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto Univ, Sakyo-ku, Kyoto 606-8502, Japan.|Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are also with Nippi Research Inst. of Biomatrix, 520-11, Kuwabara, Toride, Ibaraki, 302-0017, Japan.|Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are also with Medical Research Support Center, Graduate School of Medicine, Kyoto Univ, Yoshida Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ogura
authors.affiliation: Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are with Div. of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto Univ, Sakyo-ku, Kyoto 606-8502, Japan.|Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are also with Nippi Research Inst. of Biomatrix, 520-11, Kuwabara, Toride, Ibaraki, 302-0017, Japan.|Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are also with Medical Research Support Center, Graduate School of Medicine, Kyoto Univ, Yoshida Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Tanaka
authors.affiliation: Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are with Div. of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto Univ, Sakyo-ku, Kyoto 606-8502, Japan.|Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are also with Nippi Research Inst. of Biomatrix, 520-11, Kuwabara, Toride, Ibaraki, 302-0017, Japan.|Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are also with Medical Research Support Center, Graduate School of Medicine, Kyoto Univ, Yoshida Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: Shunji
authors.initials: S
authors.lastname: Hattori
authors.affiliation: Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are with Div. of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto Univ, Sakyo-ku, Kyoto 606-8502, Japan.|Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are also with Nippi Research Inst. of Biomatrix, 520-11, Kuwabara, Toride, Ibaraki, 302-0017, Japan.|Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are also with Medical Research Support Center, Graduate School of Medicine, Kyoto Univ, Yoshida Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Ito
authors.affiliation: Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are with Div. of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto Univ, Sakyo-ku, Kyoto 606-8502, Japan.|Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are also with Nippi Research Inst. of Biomatrix, 520-11, Kuwabara, Toride, Ibaraki, 302-0017, Japan.|Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are also with Medical Research Support Center, Graduate School of Medicine, Kyoto Univ, Yoshida Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Satoh
authors.affiliation: Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are with Div. of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto Univ, Sakyo-ku, Kyoto 606-8502, Japan.|Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are also with Nippi Research Inst. of Biomatrix, 520-11, Kuwabara, Toride, Ibaraki, 302-0017, Japan.|Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are also with Medical Research Support Center, Graduate School of Medicine, Kyoto Univ, Yoshida Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: Teisuke
authors.initials: T
authors.lastname: Takita
authors.affiliation: Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are with Div. of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto Univ, Sakyo-ku, Kyoto 606-8502, Japan.|Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are also with Nippi Research Inst. of Biomatrix, 520-11, Kuwabara, Toride, Ibaraki, 302-0017, Japan.|Authors Qian, Okada, Ogura, Tanaka, Hattori, Ito, Satoh, Takita, and Yasukawa are also with Medical Research Support Center, Graduate School of Medicine, Kyoto Univ, Yoshida Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Yasukawa
mesh_terms: D000818:Animals; D055162:Biocatalysis; D002417:Cattle; D024042:Collagen Type I / Q000737:chemistry*; D004063:Digestion; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D010434:Pepsin A / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D013379:Substrate Specificity; D013552:Swine
chemical_list: D024042:Collagen Type I; D010455:Peptides; D010434:Pepsin A
keywords: collagen; kinetic analysis; pepsin; telopeptide; variant β11