Limited pepsin digestion of bovine plasma albumin

Limited pepsin digestion of bovine plasma albumin at pH 3.7 and 25 °C in the presence of octanoic acid gave two fragments, A and B, each in about 16% yield. In the absence of octanoic acid fragment A was rapidly degraded further into smaller fragments. Sodium dodecylsulfate gel electrophoreses and amino acid analyses of fragments A and B indicated their molecular weights to be about 29,000 and 34,000, respectively. Comparative studies of the cyanogen bromide peptides of fragments A and B with those of intact albumin established that fragments A and B represent, respectively, the carboxyl and the amino terminal portions of the albumin molecule.In Tris-HCl buffer (pH 7.95) at 25 °C, fragment A has one primary binding site for octanoic acid with a binding constant about one-eighth of that of albumin. This binding constant is doubled in the presence of an equimolar amount of fragment B, although fragment B itself shows very weak activity, less than one three-hundreth of that of albumin. l- and d-tryptophans competitively bind at the same primary octanoate binding site of fragment A, just as is the case with albumin.These findings together with those of other studies suggest that the albumin molecule might consist of several compact regions and that the interactions of these regions within the molecule vary with the solvent environment and upon binding of organic ligands.     

Limited cleavage of human lactoferrin with pepsin

• 1.1. Human lactoferrin in the iron-saturated form was treated with pepsin at pH 3.0, and a fragment with a single iron-binding site was isolated in a 90% pure state.
• 2.2. Its mol. wt was near 33,000.
• 3.3. Its N-terminal residue was alanine, and its iron-binding properties were identical to those of lactoferrin.
• 4.4. The curve showing iron incorporation from lactoferrin and its fragment into reticulocytes showed a biphasic character.
• 5.5. It is concluded that lactoferrin consists of two largely independent parts, which may have arisen by a gene duplication process.

     

Fragments produced by digestion of human immunoglobulin G subclasses with pepsin in urea.

It was previously shown that digestion of human IgG1/kappa myeloma proteins with pepsin in the presence of 8 M-urea produces fragments which differ from other proteolytic fragments of IgG, including those produced by peptic digestion in aqueous buffers. The two large urea/pepsin fragments each consist of three peptides, and together account for all of the constant region of the light chains and most of the constant region of the heavy chains. Myeloma proteins of subclasses IgG2, IgG3 and IgG4 with kappa light chains were digested with pepsin in 8 M-urea, and the resulting fragments compared with those produced from IgG1/kappa proteins. Gel filtration, starch- and polyacrylamide-gel electrophoresis and sequence analysis have shown that the peptides from each subclass are analogous with those from IgG1. A brief investigation of the products of urea/pepsin digestion of myeloma proteins with lambda light chains has shown that in these proteins light-chain cleavage occurs at residue leucine-182, instead of or as well as at residue 117, where cleavage takes place in kappa chains. Comparison of sequences around sites of urea/pepsin cleavage has shown that pepsin has quite restricted specificity under these conditions.     

Limited proteolysis of bovine pepsin]

A limited proteolysis of bovine pepsin (EC 3.4.4.1) was carried out. A proteolysis-resistant C-terminal protein fragment containing about 170 amino acid residues was isolated and its N-terminal sequence was established, using Edman's automatic method. It was assumed that the fragment of bovine pepsin isolated, similar to the previosly obtained porcine pepsin fragment, is an independent constituent of the protein molecule.     

The anion-transport protein of the human erythrocyte membrane. Studies on fragments produced by pepsin digestion.

We have studied the fragmentation by pepsin in 1 M-acetic acid of the erythrocyte anion-transport protein in erythrocyte membranes. The location of the fragments obtained was determined by radioiodinating the protein with the use of lactoperoxidase, and identifying the labelled peptides obtained in peptide "maps" of thermolysin digests of the fragments. Three of the fragments were found to be related overlapping products, and shared a common C-terminus. The major site of pepsin cleavage leading to the C-termini of these fragments was shown to be close to the major site of extracellular cleavage of the protein by proteinases active at a neutral pH. Another two fragments were isolated and shown to be derived from the C-terminal portion of the protein. No well-defined large radioactive fragments of the protein were solubilized from the membrane by pepsin in 1 M-acetic acid, the bulk of the radioactivity attributable to the anion transport protein being recovered in very small fragments that could not be resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Our results suggest that the polypeptide chain of the anion-transport protein emerges at the extracellular face of the membrane 8000-13000 daltons on the N-terminal side of the major site of extracellular cleavage of the protein by proteinases that are active at a neutral pH.     

Preparation and analysis of peptide fragments produced by pepsin hydrolysis of human plasma albumin and their relationship to its structure

Human plasma albumin was prepared and subjected to proteolysis by pepsin at pH2.45 at 25 degrees for 10min. with albumin/pepsin ratio 3000:1. Five peptide fragments were detected in the proteolysate by means of zone electrophoresis and gel filtration; these were separated and purified. Molecular weights, amino acid composition and disulphide bond content of the purified fragments were determined. The results show that a high proportion of the polypeptide chain of albumin appears to have a low cystine content, and at low pH values the molecule would be expected to have a considerable degree of freedom in its structure in these regions of the chain. A tripartite model for the structure of plasma albumin is proposed.     

Isolation of albumin from whole human plasma and fractionation of albumin-depleted plasma.

The dye Cibacron Blue F-3-GA was conjugated to Sepharose to provide an affinity column for serum albumin. Passage of whole human plasma through a column of Cibacron Blue-Sepharose results in the removal of approx. 98% of the albumin. The latter can be quantitatively recovered by desorption with NaSCN. Albumin-depleted plasma can be readily resolved into discrete fractions by a combination of conventional biochemical techniques. In particular, the resolution of plasma proteins with properties similar to those of native human plasma albumin can readily be accomplished by ion-exchange chromatography of the Sepharose-dye-treated plasma on DEAE-cellulose.     

Limited hydrolysis of bovine plasma albumin at neutral and alkaline pH catalyzed by associated proteinases.

Proteinase contaminants in some plasma albumin samples have previously been shown to produce cleavage of the albumin molecule at acid pH. The F conformer, existing at pH 3.8, is cleaved near erisidue number 400 to yield a large N-terminal fragment of approximately 46,000 daltons. No cleavage was found at pH above approximately 4.4. It is shown in this paper that the proteinase contaminants are active over a broad pH range from 2.5 to 11.4 provided conditions are such as to induce some breakdown of the native conformation of the albumin molecule. Addition of Tris-borate buffer (0.1 M) at pH 7.5-9 is sufficient to permit cleavage. At pH near 9 this occurs predominantly 42,000 and 27,000 daltons. Near neutral pH substantial cleavage occurs in 4-8 M urea solution or in the presence of sodium dodecyl sulfate (AD110 complex). Under these conditions there are two large fragments (42,000 and 47,000 daltons) and essentially two small ones (20,000-27,000 daltons). Under conditions where there is no cleavage at 38-40 degrees, substantial cleavage results at 50-65 degrees but enzyme inactivation also occurs toward the top of this range. The alkaline activity is inhibited by soybean trypsin inhibitor but not by pepstatin; the reverse is true of the low pH activity. Cleavage at neutral or alkaline pH under the various conditions occurs primarily at X-Leu bonds while the low pH activity was already shown to occur at X-Phe. These facts suggest the presence of at least two enzymes. There is surprisingly little pH dependence over the range 7.5-9 in any of the media examined, even though albumin is known to undergo a significant conformational change in this range, the N leads to B transition. This transition is thought to be essentially a tertiary change with little loss of helix content. It is suggested that loss of native secondary structure, especially uncoiling of helical regions, is crucial to permit attack by these enzymes.     

Long chain fatty acid binding to human plasma albumin.

The binding of six physiologically important long chain fatty acids to defatted human plasma albumin was measured at 37 degrees in a calcium-free Krebs-Ringer phosphate buffer, pH 7.4. The data were analyzed in terms of multiple stepwise equilibria. With the saturated acids, the magnitude of the equilibrium (association) constants, Ki, increased as the chain length increased: laurate smaller than myristate smaller than palmitate smaller than stearate. Oleate was bound more tightly than stearate; by contrast, linoleate was bound less tightly than stearate. The equilibrium constants, K1 through K12, ranged from 2.4 times 10-6 - 3.5 times 10-3 m-1 for laurate to 2.6 times 10-8 - 3.5 times 10-5 m-1 for oleate. Successive values of Ki decrease for each of the acids, indicating that major cooperative binding effects do not occur over the physiological range of fatty acid concentrations. In no case could the Ki be segregated into distinct classes, suggesting that any grouping of albumin binding sites is somewhat arbitrary. The results were inconclusive concerning whether premicellar association of unbound fatty acid occurs. Although corrections for premicellar association produced very little change in the Ki values for myristate, they raised the Ki for palmitate and stearate by 300 to 700 per cent. A sigmoidal relationship was obtained when the logarithm of Ki was plotted against chain length for the saturated fatty acids containing 6 to 18 carbon atoms, indicating that the binding energy is not simply a statistical process dependent only on the fatty acid chain length. This selectivity that albumin contributes to the binding process may be due to varying degrees of configurational adaptability of its binding sites as the fatty acid increases in length.     

Structure of N-terminal cyanogen bromide fragment of human plasma albumin.

The complete amino acid sequence of 87 residues of cyanogen bromide fragment CB1 (Asp), the N-terminal fragment of human plasma albumine molecule, has been established. The sequence was determined from the characterization of all tryptic peptides and of chymotryptic arginine-containing peptides in the fragment digested. Overlaps were obtained by tryptic and chymotryptic cleavage of the maleylated S-sulfo derivative of fragment CB1(Asp). Residue 34 is the only cysteine residue in the albumin molecule and it was determined in the form of S-carboxymethyl-cysteine. Edman and dansyl-Edman degradation were used for the sequential analysis.     

Limited nuclease digestion of heterogeneous nuclear ribonucleoprotein in nuclei.

We have used limited nuclease digestion of nuclei to probe the structure of nuclear ribonucleoprotein (nRNP). Analysis of [³H]uridine-labeled heterogeneous nuclear RNA isolated from nuclease digested nuclei revealed preferential generation of discrete bands of RNA ranging in size from 1.5 × 10⁵ to 6 × 10⁵ daltons. The nuclease digestion pattern of nRNP differed from the nuclease digestion pattern obtained with chromatin in that the RNA bands generated in these experiments were transient, appearing only early in the course of digestion, and no stable nRNP monomer size was evident. Therefore, although nRNP may be organized in a regular configuration, nRNP structure differs considerably from the repeating subunit structure of chromatin.     

Neurotensin-like immunoreactivity generated by pepsin from human plasma and gastric tissue

The present study demonstrates precursors of neurotensin-like immunoreactivity (NTLI) endogenous to human gastric tissue and plasma, and the existence of a gastric NTLI-generating enzyme system. The molecular size of the NTLI-precursors in plasma and gastric tissue were estimated by gel permeation chromatography to be ca 50,000-60,000 and 60,000-70,000 Da, respectively. The neurotensin-like peptide generated from the precursor was detected with a carboxyl-terminally directed antiserum but did not cross-react with an amino-terminally directed antiserum. A neurotensin-like peptide isolated from pepsin-treated human plasma was characterized by mass spectrometry and its amino acid sequence determined. This novel nonapeptide, referred to as kinetensin, failed to affect pentagastrin-stimulated acid secretion or blood pressure in the rat. Sequence homologies between neurotensin, kinetensin and proteins of the serum albumin family suggest a common evolutionary origin and raise questions regarding albumin-like proteins as precursors of regulatory peptides.