Insulins bovine

Sanger also determined the sequence of the A chain and identified the cysteine residues involved m disulfide bonds befween fhe A and B chains as well as m fhe disulfide linkage wifhin fhe A chain The complefe insulin sfruefure is shown m Figure 27 11 The sfruefure shown is fhaf of bovine insulin (from cattle) The A chains of human insulin and bovine insulin differ m only fwo ammo acid residues fheir B chains are identical except for the ammo acid at the C terminus... 

Biosynthetic Human Insulin from E. coli. Insulin [9004-10-8] a polypeptide hormone, stimulates anaboHc reactions for carbohydrates, proteins, and fats thereby producing a lowered blood glucose level. Porcine insulin [12584-58-6] and bovine insulin [11070-73-8] were used to treat diabetes prior to the availabiHty of human insulin [11061 -68-0]. AH three insulins are similar in amino acid sequence. EH LiHy s human insulin was approved for testing in humans in 1980 by the U.S. EDA and was placed on the market by 1982 (11,12). 

FIGURE 5.17 The hormone insulin consists of two polypeptide chains, A and B, held together by two disulfide cross-bridges (S—S). The A chain has 21 amino acid residues and an intrachain disulfide the B polypeptide contains 30 amino acids. The sequence shown is for bovine insulin. 

The diabetic rats were treated with 18 IU of bovine insulin imbibed into polyacid resins b.i.d. orally using 1 cc syringes and gavage tubes. After 14 days of treatment the rats were sacrificed about 1.5 hours after the last dose. Blood samples were taken and assayed for immunoactive insulin activity (Amersham-Searie RIA kit) and serum glucose levels (glucose oxidase colorimetric assay, Sigma 510 Glucose Kit). 

Diabetic Rats-Phase II. This protocol was similar to that in phase I except that 65 mg/kg STZ was employed, 54 HJ bovine insulin in the polyacid resin was utilized in each dose and the treatment period was for nine days. Groups of rats (four per group) were then sacrificed at 30,90,165 and 255 minutes after the last dosing. 

HPLC reverse phase procedures were established to follow the continuous release rates of a variety of agents from the two resins. Also a USP standard release test procedure (8) was used. Because of its ease of detection at the higher ultraviolet wave lengths, bovine insulin was used as the model delivery agent... 

PROTECTING albumin Bovine Insulin Dipeptide Acid ... 

Figure 11. Serum assays of diabetic rats after 14 days of treatment with 18 IU of bovine insulin, b.i.d., in polyacrylic and poly acrylic acid resins.

When diabetic rabbits (24) were treated with 50 IU of bovine insulin imbibed at 50 mg/g poly (acrylic acid) (Figure 14) no reduction in serum glucose over that achieved by the dry blend control could be detected. Pretreatment of the animals with oral doses of either a penetration enhancer, sodium taurocholate, or a protease inhibitor, aproteinin, failed to improve the insulin activity. One possible explanation for this unexpected lack of activity might be that the diseased animals exhibit impaired ileal absorption of fluids (25). 

Figure 13. Normalized reduction of serum glucose in normal rabbits by oral administration of bovine insulin imbibed into a poly(acrylic acid) matrix.

Online LC-ESI-TOF-MS experiments are carried out in a very similar fashion to the off-line NPS-HPLC separations described above, with a few notable exceptions. Firstly, 0.3% (v/v) formic acid is added to each mobile phase to counteract the ionization suppression induced by TFA. Because of the formic acid UV detection must be carried out at 280 nm (as opposed to 214 nm). To aid in normalization between runs 1 jag of Bovine insulin (MW = 5734 Da) is added to each chromatofocusing fraction prior to injection onto the column. Finally, the flow is split postcolumn directing 200 JlL/min into the ion source and the remaining 300 JlL/min through the UV detector and fraction collection. 

As described above all samples were separated online using LCT ESI-TOF-MS then normalized for relative quantitation using a bovine insulin internal standard. Fractions were then collected for MAFDI-TOF-MS PMF, digested with modified porcine trypsin, and analyzed using the TofSpec2E. Following this analysis, three major classes of differentially expressed including proteins were revealed in these... 

Immobilized dihydrolipoamide (thioctic acid) (Gorecki and Patchornick, 1973 Gorecki and Patchornick, 1975) and immobilized N-acetyl-homocysteine thiolactone (Eldjarn and Jellum, 1963 Jellum, 1964) are the two most commonly used immobilized disulfide reductants. In addition, immobilized TCEP provides a reducing matrix that is free of thiols (Thermo Fisher). Such immobilized reductants successfully can be used to reduce many types of biological disulfides, including small molecules like oxidized glutathione and bovine insulin. They... 

Mature insulin consists of two polypeptide chains connected by two interchain disulfide linkages. The A-chain contains 21 amino acids, whereas the larger B-chain is composed of 30 residues. Insulins from various species conform to this basic structure, while varying slightly in their amino acid sequence. Porcine insulin (5777 Da) varies from the human form (5807 Da) by a single amino acid, whereas bovine insulin (5733 Da) differs by three residues. 

Boundary managing, in R D, 21 619-620 Boundary spanning, in R D, 21 619 Bound chloride formation, 10 358 Bound moisture, 9 96 Bourdon tube, 20 647-649 Boussinesq approximation, 11 779 Boutique fuels, 12 419 Bovatec, 20 136 Bovine hemoglobin, 4 125 Bovine insulin, 3 817 Bovine serum albumin (BSA), 20 573 properties of standard, 3 836t Bovine somatotropin (BST), 10 871 Bovine spongiform encephalitis/... 

Use of the anionic CP allowed for detection of amyloid fibril formation in both bovine insulin and chicken lysozyme proteins (Fig. 16). The polymer in buffer... 

Fig. 16 (a) Description of the detection of amyloid fibrils in proteins with an anionic conjugated polymer, PTAA. (b) Emission spectra (bottom) of PTAA-Native bovine insulin (filled square) and PTAA-amyloid fibrillar bovine insulin (x). (c) Kinetics of insulin amyloid fibril formation monitored by PTAA fluorescence [29]... 

Fig. 17 (a) Chemical structure of polythiophene poly((3,3"-di[(S)-5-amino-5-carbonyl-3-oxapen-tyl]-[2,2 5 2"])-5-,5"-terthiophenylene hydrochloride), PONT, (b) Emission spectra of 6.5 pM PONT—HC1 (on a chain basis) in 25 mM HC1 (black spectrum), 25 mM HC1 with 5.0 pM of native bovine insulin (blue spectrum), 25 mM HC1 with 5.0 pM fibrillar bovine insulin (red spectrum). The emission spectra were recorded with excitation at 400 nm [31]... 

Materials Required Solution (1) Dissolve 10 mg of insulin in 1 ml of the mobile phase Solution (2) Dilute 100 i/ of solution (1) to 10 ml with the mobile phase and Solution (3) Dissolve 10 mg of procine insulin EPCRS of bovine insulin EPCRS, as appropriate, in 1 ml of the mobile phase. 

With methods for the quantitative analysis of amino acids to hand, the way was now open for the determination of amino acid sequences. Purified bovine insulin was relatively freely available. On the basis of ultracentrifugal analysis (Gutfreund and Ogston), a molecular weight of 12,000 was assumed—as it later emerged, a factor of 2 too high. One of the advantages from the choice of insulin as the protein to sequence was that tryptophan is absent. A 100% recovery of the amino acids could therefore be obtained easily by simple hydrolysis with HC1. In 1948 Tristram reported the complete amino acid composition of the protein. 

Insulin was originally (since the 1930s) obtained from porcine and bovine extracts. Bovine insulin differs from human insulin by three amino acids, and it can elicit an antibody response that reduces its effectiveness. Porcine insulin, however, differs in only one amino acid. An enzymatic process can yield insulin identical to the human form. Currently, insulin is produced via the rDNA process it was the first recombinant biopharmaceutical approved by the FDA in 1982. The recombinant insulin removes the reliance on animal sources of insulin and ensures that reliable and consistent insulin is manufactured under controlled manufacturing processes. A description of diabetes meUitus and insulin is presented in Exhibit 4.13. 

Fig. 3.27. The isotopic pattern calculated for [M+H]" of bovine insulin at different resolutions (Rio%). Note that the envelope at R = 1000 is wider than the real isotopic pattern.

Fig. 4.7. The molecular ion signal of Ceo at m/z 720 (a) and the quasimolecular ion signal of bovine insulin, m/z 5734.6, (b) as obtained on a TOF instrument in linear mode (left) and reflector mode (right). All other experimental parameters remained unchanged.

Californium plasma desorption ( Cf-PD) dates back to 1973 [4-6,22,154-156] and was the first method to yield quasimolecular ions of bovine insulin. [157] Practically, Cf-PD served for protein characterization, a field of application which is now almost fully transferred to MALDI or ESI (Chaps. 10,11). [158]... 

Example The Cf-PD mass spectrum of bovine insulin exhibits the [M+H]" quasimolecular ion as well as the doubly charged [M+2H] and triply charged [M+3H] ion (Fig. 9.20). [156] Fragment ions corresponding to the A and B chain as well as some a-type peptide fragments ions are observed in addition. 

AAm monomer was purchased from Junsei Chemical Co., Japan. DMAEMA monomer, ammonium persulfate (APS), and tetramethylethyl-diamine (TEMED) were purchased from Aldrich. Bovine insulin, N,N-azobis(isobutyronitrile) (AIBN) and glucose oxidase (GOD) were purchased from Sigma Chemical Co. DMAEMA monomer was distilled before use. Other reagents were used as received. 

Lyophilized copolymer was ground down to colloidal dimensions (<1 iJim) using a laboratory planetary mill (Pulverisette, Fritsch GmbH). A 110-mg sample of copolymer powder, 20 mg of bovine insulin, and 20 mg of GOD were mixed, and the mixture was compressed into a disk-shaped matrix of 5-mm thickness and 15-mm diameter. 

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