The Chromatography of Insulin on Deae-Cellulose in Buffers Containing 8m Urea

Thompson, Eop; O'donnell, IJ

doi:10.1071/bi9600393

Cited by 24 publications

(9 citation statements)

References 13 publications

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“…However, improved analytical methods showed that crystallized insulin included proinsulin and intermediates, nonconvertible insulin dimers, arginyl and ethyl insulin, deamidated insulin, monodesamido insulin, and various noninsulin peptides (7)(8)(9). RIAs done in 1982 showed that twice-crystallized Novo monocomponent insulin contained proinsulin and its intermediates as well as trace amounts of other peptide hormones: proinsulin-like immunoreactivity up to 20,000 ppm, glucagon 45 ppm, pancreatic polypeptide 43 ppm, somatostatin 1.4 ppm, and vasoactive intestinal peptide 0.3 ppm (10).…”

Section: A Animal Insulin Preparationsmentioning

confidence: 99%

Immunological Responses to Exogenous Insulin

et al. 2007

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Regardless of purity and origin, therapeutic insulins continue to be immunogenic in humans. However, severe immunological complications occur rarely, and less severe events affect a small minority of patients. Insulin autoantibodies (IAAs) may be detectable in insulin-naive individuals who have a high likelihood of developing type 1 diabetes or in patients who have had viral disorders, have been treated with various drugs, or have autoimmune disorders or paraneoplastic syndromes. This suggests that under certain circumstances, immune tolerance to insulin can be overcome. Factors that can lead to more or less susceptibility to humoral responses to exogenous insulin include the recipient's immune response genes, age, the presence of sufficient circulating autologous insulin, and the site of insulin delivery. Little proof exists, however, that the development of insulin antibodies (IAs) to exogenous insulin therapy affects integrated glucose control, insulin dose requirements, and incidence of hypoglycemia, or contributes to beta-cell failure or to long-term complications of diabetes. Studies in which pregnant women with diabetes were monitored for glycemic control argue against a connection between IAs and fetal risk. Although studies have shown increased levels of immune complexes in patients with diabetic microangiopathic complications, these immune complexes often do not contain insulin or IAs, and insulin administration does not contribute to their formation. The majority of studies have shown no relationship between IAs and diabetic angiopathic complications, including nephropathy, retinopathy, and neuropathy. With the advent of novel insulin formulations and delivery systems, such as insulin pumps and inhaled insulin, examination of these issues is increasingly relevant.

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Section: A Animal Insulin Preparationsmentioning

confidence: 99%

Immunological Responses to Exogenous Insulin

et al. 2007

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“…The available data (Moore and Stein 1956;Hill, Kimmel, and Smith 1959;Cole 1960;Thompson and O'Donnell 1960) indicate that most successful chromatographic separations of proteins with similar isoelectric points have been achieved at a pH near the isoelectric points of the proteins. Although the conditions under which gluten is isoelectric are ill-defined (pH 5-7), greater resolution would be expected at the pH of this study, 4•1, than at the values 3•4-1•5 used by Woychik, Dimler, and Senti (1960).…”

Section: Discussionmentioning

confidence: 99%

Chromatographic Fractionation of the Acetic Acid Soluble Proteins of Wheat Flour on Carboxymethyl-Cellulose

Simmond¹,

Winzor²

1961

Aust. Jnl. Of Bio. Sci.

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The acetic acid soluble proteins extracted from wheat flour have been separated into 11 fractions by a chromatographic procedure using columns of carboxymethyl- cellulose, equilibrated with O� 005M acetate buffer, pH 4�1. Under theso conditions one fraction passes unretarded through the column. Eight further fractions are eluted at pH 4� 1 by employing a gradient to O� 2M N aCl in the presence of 1M dimethyl formamide (DMF) which prevents protein precipitation at ionic strengths greater than 0�03.

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“…The separated chains may be contaminated with each other, with incompletely reduced and carboxymethylated insulin or with contaminant proteins other than insulin. Studies of commercial insulin preparations by countercurrent distribution (Harfenist and Craig 1952), partition chromatography (Porter 1953;Chrambach and Carpenter 1960), or ionexchange chromatography (Boardman 1955;Cole 1960;Thompson and O'Donnell 1960) have shown that only minor amounts of proteins other than insulin are present but that partially deamidated forms of insulin are often present (Harfenist and Craig 1952;Harfenist 1953;Sundby 1962;Slobin and Carpenter 1963;Carpenter and Hayes 1963). The insulin preparations from Commonwealth Serum Laboratories used in this study are the purest examined in this laboratory (Human and Leach 1961;Thompson and O'Donnell 1960).…”

Section: Resultsmentioning

confidence: 99%

The Preparation of the A and B Chains from Reduced and S-Carboxymethylated Beef Insulin

Thompson¹,

O'donnell²

1966

Aust. Jnl. Of Bio. Sci.

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SummaryThe A and B chains of reduced and S-carboxymethylated insulin have been prepared in a pure state by two procedures.The chains could be separated readily in almost pure form by chromatography on DEAE-cellulose in buffers containing 8M urea, or by pH fractionation in the presence of O' 5M potassium chloride. These fractions were then freed from contaminating traces of each other and other impurities by gel-filtration on Sephadex . G-50 in buffers containing 8M urea. The S-carboxymethylated B chain could also be purified on Sephadex G-50 using 50% formic acid. The retention of these polypeptides by Visking 18/32 cellulose dialysis tubing greatly facilitated their isolation.The rate of carbamylation of the B chain of S-carboxymethylated insulin by the cyanate formed in urea solutions has been studied. In agreement with Cole (1961) and Stark (1965) carbamylation at about pH 7·5 was found to involve only the a-amino group. At pH 10 it was found that there was negligible carbamylation of either the a-amino or the €-amino groups.There was no evidence of peptide bond breakage when the S-carboxymethylated B chain was allowed to stand at room temperature in 50% formic acid for 24 hr.

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The Chromatography of Insulin on Deae-Cellulose in Buffers Containing 8m Urea

Cited by 24 publications

References 13 publications

Immunological Responses to Exogenous Insulin

Immunological Responses to Exogenous Insulin

Chromatographic Fractionation of the Acetic Acid Soluble Proteins of Wheat Flour on Carboxymethyl-Cellulose

The Preparation of the A and B Chains from Reduced and S-Carboxymethylated Beef Insulin

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