Date of Award
1974
Degree Type
Dissertation
Degree Name
Doctor of Philosophy in Biological Sciences
Department
Biological Sciences
First Advisor
C.O. Chichester
Abstract
The effects of the Maillard reaction on the biological activity of insulin as a hormone, and on the utilization of egg albumin as a source of amino acids have been studied.
In order to know the number, the position, and the effect of the glucose residues that bind a protein, crystalline zinc insulin was stored with 14C-glucose at 37° and 68% R.H. for four months. The unreacted sugar was separated from insulin in a Bio-Gel column and the bound radioactivity counted. The first sugar residue reacted within five days, and additional 2.7 residues reacted through the end of the storage. Binding of 1.4 residues increased the acid solubility of insulin ten times suggesting impairment of hexamer formation. The ability of this Maillard insulin in controlling the blood glucose and tryptophan levels suffered little or no change. After four months the s9lubility decreased by a factor of 1000.
Since the α-amino groups are more reactive than any other group in a protein, the N-terminal Maillard reaction was studied using two dipeptides. The reaction of glycyl-L-leucine with an excess of glucose gave a compound which, after chromatographic purification, was tentatively identified as N, N-di(l-deoxy-2-ketosyl)-glycyl-L-leucine. This Maillard product was not hydrolyzed by leucine-amino peptidase. In vivo, the compound was completely unabsorbed from the intestine of duodenum/portal vein cannulated rats, indicating that the residue adjacent to the N-terminal amino acid of this Maillard dipeptide was not released and transported even if it did not directly participate in the Maillard reaction. From the portal blood levels of free tryptophan, it was learned that the various N-substituted dipeptides present in an L-lysyl-L-tryptophan premelanoidin mixture may be hydrolyzable and serve as a source of tryptophan without interfering with the absorption of the normal dipeptide.
The exclusion of small, soluble peptides from absorption suggested that they may be detected in the feces. The soluble fraction of the feces of rats fed Maillard egg albumin was analyzed, after extraction and filtration with solutions of (i) sodium chloride, (ii) ammonium acetate, and (iii) pyridine acetate. The NaCl extracts showed the accumulation of ninhydrin-positive bands of about (I) 1300, (II) 1000, and (III) 800 molecular weight, all exhibiting browning characteristics. The first two bands contained high proportions of lysine, plus other essential and non-essential amino acids. Band III yielded 90% NH3 upon hydrolysis. (ii) Portions from the ammonium acetate extracts, containing bands I and II, were resolved into six peaks by cation exchange and analyzed for amino acids. (iii) Samples treated with pyridine acetate gave a profile different from that in (1). Cation exchange chromatography revealed some fifteen peaks, which were neither peptides nor glycosidic residues. Maillard ovalbumin tagged with either 14C-lysine or -tryptophan was fed to rats to assess the fecal recovery. Feeding the Maillard ovalbumin raised the recovery of radioactivity from 1 to 7% for the former, and from .2 to 3% for the latter. Such loss was not considered significant, and thus other possibilities are suggested to account for the drastic reduction of nutritive value of mildly brown egg albumin.
Recommended Citation
Amaya-Farfan, Jaime, "Biological Inactivation of Proteins by the Maillard Reaction" (1974). Open Access Dissertations. Paper 685.
https://digitalcommons.uri.edu/oa_diss/685
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