A Study of Surface Physical Properties of New Surfactant Using Synthetic Peptides of Surfactant Protein-B

PURPOSE: To produce a new generation of artificial pulmonary surfactant(PS), surfactant protein (SP)-B from human PSwas isolated, and the amino acid sequences of these proteins were studied. Artificial peptides of human SP-B were synthesized. New artificial PS preparations which were cornposed of phospholopids and two artificial synthetic SP-B peptides were made, and the surface physical properties of these new PS preparations were tested. METHODS: The purities of SP-B were assessed by SDS-polyacrylamide gel and the amino acid sequences of these proteins were determined. We synthetized two peptides SP-1 and SP-2 and the amino acid sequences were as follows,' SP-1: RMLPQLVCRLVLRCSMD, SP-2: RMLP- QLVCRLVLRCSM. Surface physical properties of newly artificial PSs, which were composed of a mixture of phospholipid(PL) and SP-1 or SP-2(sample A; PL+SP-1, sample B; PL+SP-2), were measured by surface spreading, adsorption rate, and surface tension-area diagram. RESULTS: The amino acid sequence of human SP-B was obtained. We produced the artificial peptides of SP-B and prepared the new generation PS(sample A and sample B). The order of the superiority of spreading and adsorption rate was Surfacten

Purification and Analysis of Amino Acid Sequences of Pulmonary Surfactant Proteins

PURPOSE: For the synthesis of surfactant protein(SP) peptides and production of next generation artificial pulmonary surfactant(PS), we have isolated SP-B, C from bovine PS, and studied the biochemical properties and amino acid sequences of these protein-peptides. METHODS: Crude surfactant and purified surfactant were isolated from materials extracted from the bovine lung lavage. The hydrophobic SP-B, C were purified by Sephadex LH 60 column chromatography from PS. The purities of SP-B, C were assessed by tricine buffer SDS-polyacrylamide gel electrophoresis and the amino acid sequences of these proteins were determined using Beckman PI-2090. RESULTS: The molecular weights of SP-B, C shown in SDS-polyacrylamide gel electrophoresis were as follows; 15,000-18,000(oligomer) Da for SP-B, 3,500-5,000 Da for SP-C. The amino acid sequences were; FPIPLPYCWL LRTLIKKIQA VIPKGVLAMT VAQCHVVPL LVGGIQQLV IEYSVILLTD TLLGRLPNLV CGLRLRCSG in SP-B, LIPCCPVNIK RLLIVVVVVV LLVVVIVGAL LMGL in SP-C, respectively. These results indicated that the amino acid sequences of bovine SPs were different. CONCLUSION: The SP-B, C were purified from bovine PS, and amino acid sequences of SP-B, C were determined. Further studies are needed for the development and use of next generations of exogenous PS preparation based on synthetic SP-peptides for the treatment of neonatal RDS in the future.

Preparation and in vitro Physical Activities of Crude Natural Surfactant and Artificial Pulmonary Surfactant Containing Synthetic Peptide and Phospholipid Mixtures

PURPOSE: In this study, natural pulmonary surfactant was extracted from bovine lung lavage and its surface activity was determined. To investigate the usefulness of synthetic peptides reconstituted with phospholipid as artificial surfactant, truncated peptides from surfactant protein (SP)-B were synthesized and restored the surface tension lowering activities when appropriately recombined with phospholipid. METHODS: Crude natural surfactant (CNS) was isolated from lung lavage by centrifugation and organic solvent for the extraction of pulmonary surfactant was selected to satisfy the in vitro physical properties. Two truncated peptides derived from C-terminal end of bovine SP-B hydrophobic protein were selected and synthesized. To prepare artificial surfactant, synthetic peptides was added to the phospholipid mixture. The various surfactant mixtures were assayed for in vitro physical activity with the Wilhemly plate method and were determined by surface spreading rate, surface adsorption rate and surface tension-area diagram. RESULTS: CNS-chloroform methanol (CM) displayed efficient surface activity compared with clinically used Surfacten but CNS-BuOH did not. The artificial surfactants containing phospholipid mixture and synthetic peptide were analyzed for their surface activities and displayed significant surfactant properties. CONCLUSION: 1-Butanol or CM (3:1) was used as an extraction solvent for CNS. CNS-CM showed more efficient surface activity than CNS-BuOH. Two synthetic peptides composing artificial pulmonary surfactant were designed and mixing ratio of peptide and phospholipid was established. Artificial surfactant dispalyed weaker surface activity than natural surfactant but significant surfactant activity.

Surfactant Protein SP-A, B, and C: Purification and Biochemical Characterization

PURPOSE: Several kinds of exogenous pulmonary surfactants (SF), either synthetic or animal- derived, are being used for the replacement therapy in respiratory distress syndrome (RDS) of newborn, especially in premature infants, and improved the neonatal mortality and morbidity. Because synthetic preparations are lack of surfactant protein (SP) and animal-derived preparations cause immunogenecity of heterogenous SP, there have been great necessity for the development of next generation of exogenous SF which made by new technology to produce new type of human SF (contained human synthetic SP). There are two methods to make next generation of SF (mixtures of phospholipids and human synthetic SP) which are using of recombinant SP or synthetic peptides of SP. For the synthesis of SP peptides and production of next generation of SF, at first step, we have isolated SP-A, B, and C from bovine lung SF, and studied the biochemical properties of these proteins. METHODS: Crude natural surfactant (CNS) and purified natural surfactant (PNS) were isolated from materials which extracted from the bovine lung lavage. The hydrophilic SP-A was purified from PNS by method of modified Hawgood, and hydrophobic SP-B, C were purified by Sephadex LH 60 column chromatography. The purities of the purified SP-A and SP-B, C were assessed by 12% SDS-polyacrylamide gel and tricine buffer SDS-polyacrylamide gel, respectively and the N-terminal amino acid sequences of these proteins were determined using Beckman PI-2090. The polyclonal anti-serum against SP-A was prepared by immunization of the purified SP-A into the mouse and the immunization of the purified SP-A into the mouse and the immunogenecity of SP-A was confirmed by indirect ELISA. RESULTS: Total 22 gm of CNS, 11 gm of PNS, and 2.5 mg of SP-B and 3.2 mg of SP-C/ 1 gm of CNS, were purified from one bovine both lungs. The molecular weights of SP-A, B, C shown in SDS-polyacrylamide gel were as follows; 28,000-35,000 Da (molecular weight) of SP-A, 15,000-18,000 Da of SP-B, 3,500-5,000 Da of SP-C. The partial N-terminal amino acid sequences of each SPs were; Leu-Glu-His-Asp-Val-Lys- Glu-Val-.... in SP-A, Phe-Pro-Ile-Pro-Ile-Pro-Tyr-.... in SP-B, Leu-Ile-Pro-.... in SP-C, respectively. These results indicated that the amino acid sequences of bovine SPs were different from those of other species, i.e., human, dog and rat, which were reported previously by another investigators and species-specific patterns were shown. The immunogenecity of the purified SP-A was confirmed by the production of polyclonal antibody against mouse. The polyclonal antibody of SP-A could be used for measuring the amount of pulmonary SF in lung lavages. Carbohydrate portion of SP-A was cleaved with N-glycocisidase F. This result suggested that carbohydrate group could be N-glycosylated in some arginine residue of SP-A. CONCLUSIONS: The SP-A, B, C were purified from bovine lung SF, and N-terminal amino acid sequences of each SP-A, B, C were determined. Further studies were needed for the development and use of next generations of exogenous SF preparation, which based on synthetic SP-peptides, for the treatment of neonatal RDS in the future.