Harold B. White

Long-Range Order in Biomembranes

Publisher Summary The central problem of membrane structure and its correlation with physiological and biochemical functions is to define the organization of constituent molecules. The existence of bilayers in biomembranes is established by a variety of physicochemical techniques. It has been shown that the subtleties of organizational and phase characteristics of the bilayer arise from the segmental motion and the transverse, rotational, and lateral mobilities of constituent lipids. These molecular features of lipids in the bilayer organization account for dielectric, viscoelastic, partitioning, and passive permeability characteristics. Experimental evidence indicates that the membrane lipids not only create a barrier to the free entry and exit of molecules into and out of the cell, but lipids also provide a matrix in/on which biochemical reactions can take place; through which certain metabolites can pass selectively; and with which recognition, adhesion, aggregation and fusion of cells can be mediated.

A Sulfhydryl Oxidase from Chicken Egg White

A dimeric glycoprotein containing one FAD per ∼80,000 Mr subunit has been isolated from chicken egg white and found to have sulfhydryl oxidase activity with a range of small molecular weight thiols. Dithiothreitol was the best substrate of those tested, with a turnover number of 1030/min, a Km of 150 μM, and a pH optimum of about 7.5. Oxidation of thiol substrates generates hydrogen peroxide in aerobic solution. Anaerobically, the ferricenium ion is a facile alternative electron acceptor. Reduction of the oxidase with dithionite or dithiothreitol under anaerobic conditions yields a two-electron intermediate (EH2) showing a charge transfer band (λmax 560 nm; εobs 2.5 mM−1 cm−1). Complete bleaching of the flavin and discharge of the charge transfer complex require a total of four electrons. Borohydride and catalytic photoreduction give the same spectral changes. EH2, but not the oxidized enzyme, is inactivated by iodoacetamide with alkylation of 2.7 cysteine residues/subunit. These data indicate that the oxidase contains a redox-active disulfide bridge generating a thiolate to oxidized flavin charge transfer complex at the EH2 level. Sulfite treatment does not form the expected flavin adduct with the native enzyme but cleaves the active site disulfide, yielding an air-stable EH2-like species. The close functional resemblance of the oxidase to the pyridine nucleotide-dependent disulfide oxidoreductase family is discussed.

Messenger RNA Structure: Compatibility of Hairpin Loops with Protein Sequence

Examination of the amino acid sequences of human cytochrome c and the α-chain variant of human hemoglobin Constant Spring has revealed the possiblity for base-paired hairpin loops in the messenger RNAs for these proteins. A similar analysis of the bacteriophage R17 coat protein suggests an additional unobserved loop in the R17 RNA. If such loops are present in messenger RNAs generally, it would suggest that DNA has more than one stable base-paired conformation.

What skills should students of undergraduate biochemistry and molecular biology programs have upon graduation

Biochemistry and molecular biology (BMB) students should demonstrate proficiency in the foundational concepts of the discipline and possess the skills needed to practice as professionals. To ascertain the skills that should be required, groups of BMB educators met in several focused workshops to discuss the expectations with the ultimate goal of clearly articulating the skills required. The results of these discussions highlight the critical importance of experimental, mathematical, and interpersonal skills including collaboration, teamwork, safety, and ethics. The groups also found experimental design, data interpretation and analysiand the ability to communicate findings to diverse audience to be essential skills. To aid in the development of appropriate assessments these skills are grouped into three categories, 1) Process of Science, 2) Communication and Comprehension of Science, and 3) Community of Practice Aspects of Science. Finally, the groups worked to align these competencies with the best practices in both teaching and in skills assessment.

Phosphorylation sites in riboflavin-binding protein characterized by fast atom bombardment mass spectrometry

The capability of fast atom bombardment mass spectrometry for characterization of phosphorylation sites in a tryptic peptide from chicken egg yolk riboflavin-binding protein has been evaluated. The quality of information about molecular weight, amino acid sequence, phosphorylation sites, and microheterogeneity is evaluated as a function of the sign of the ions analyzed, the nature of the counter ions associated with the phosphate substituents, sample matrix, and various instrumental parameters. The intact octaphosphorylated 23-residue peptide was found to be susceptible to mass spectral analysis. Information from the negative ion spectrum was used in conjunction with complete sequence information and experiments which showed that all phosphates were attached to serine residues. Phosphorylated and unphosphorylated serine residues were identified and the sample was shown to be homogeneously octaphosphorylated.

Competitive binding assays for riboflavin and riboflavin-binding protein.

Abstract A competitive binding procedure that can be used to determine either riboflavin or riboflavin-binding protein has been developed. Riboflavin-binding protein from chicken egg white binds tightly to DEAE-cellulose while free riboflavin does not. Stock [2-14C]riboflavin solutions, diluted with varying amounts of a standard unlabeled riboflavin solution or an unknown sample, are mixed with aporiboflavin-binding protein and washed through small DEAE-cellulose columns. The protein-bound riboflavin is batch eluted into scintillation vials, counted, and the unknown samples compared to a standard curve. This is a simple, rapid method for assaying riboflavin by isotope dilution. By a slight modification of the incubation conditions of this procedure, the degree of saturation and amount of riboflavin-binding protein can be determined. Data from both assays can be represented by linear plots in which slopes or intercepts correspond to unknown values. The principles presented here have been extended to the assay of biotin and avidin and should apply to other vitamins and vitamin-binding proteins.

Carbohydrate compositional effects on tissue distribution of chicken riboflavin-binding protein

Riboflavin-binding proteins (RBP) purified from chicken egg white, yolk and the serum of laying hens differ in their carbohydrate compositions reflecting tissue-specific modifications of a single gene product. All three are complex glycoproteins having more than twice as many N-acetylglucosamine residues (greater than 12) as mannose residues (approx. 6). Egg white RBP is distinctive in having only one sialic acid and two galactose residues. Serum RBP contains approx. five sialic acid and seven galactose residues. In addition there is one residue of fucose. The carbohydrate composition of yolk RBP indicated the hydrolysis, respectively, of one, one, two and 3 residues of sialic acid, fucose, galactose, and N-acetylglucosamine from its precursor, serum RBP. The effect of these differing levels of glycosylation on plasma clearance, ovarian uptake and tissue distribution of 125I-labeled riboflavin-binding proteins in laying hens were compared. 2 h after intravenous injection, 19% of the egg white RBP, 29% of the yolk RBP, and 37% of the serum RBP remained in circulation. The kinetics of plasma clearance was distinctly biphasic for each of the radioiodinated proteins. The initial rapid-turnover component (t1/2 = 13 min) ranged from 27% of the serum RBP sample to 48% of the egg white RBP sample. The remaining slow-turnover components were cleared with half-lives of 81 min (egg white RBP), 101 min (yolk RBP), and 121 min (serum RBP). 16 h after injection, only 4% of the egg white RBP was deposited in the yolk of developing oocytes while about 12% of the serum RBP and yolk RBP was deposited. This highly significant difference is apparently due to preferential, carbohydrate-dependent clearance of egg white RBP by the liver rather than preferential uptake of serum and yolk RBP by the ovarian follicle. We find no evidence for carbohydrate-directed uptake of riboflavin-binding protein by the ovarian follicle.

[36] Isolation of avian riboflavin-binding protein

Publisher Summary This chapter describes the isolation of avian riboflavin-binding protein (RBP). As avian riboflavin binding protein (RBP) is highly anionic, therefore purification by conventional ion-exchange chromatography is quite effective. The chapter considers the isolation of RBP from egg white, yolk, and hen plasma. The purification of egg white riboflavin-binding protein relies on the affinity of RBP for diethylaminoethyl (DEAE)-cellulose under conditions in which most other proteins do not bind. Egg white RBP can be purified to apparent homogeneity in three steps—namely, batch adsorption to DEAE-cellulose, salt precipitation, and chromatography on CM- or DEAE-cellulose. The strong absorbance of protein-bound riboflavin at 455 nm may be used as a visual indicator for the holoprotein, throughout the purification procedures. As the properties of yolk RBP are similar to those of its egg white counterpart, much of the isolation procedure is identical. The major difference is in the initial preparation of the yolk prior to DEAE batch treatment. The purified preparations of RBP should be examined for homogeneity and assayed for riboflavin-binding capacity. Pure egg white RBP migrates as a single band during electrophoresis on sodium dodecyl sulfate (SDS)–polyacrylamide gels with an apparent molecular weight of 34,000–36,000.

Biotin‐binding Proteins and Biotin Transport to Oocytes

The eggs of chickens and other birds contain two proteins that bind biotin. Both are homotetrameric proteins of similar size. In contrast to the well-characterized egg white avidin, egg yolk biotin-binding protein has a very acidic isoelectric point, binds biotin with lower affinity, and is usually saturated with biotin. Like other egg yolk proteins, biotin-binding protein appears to be synthesized in the liver, transported by the blood stream to the ovary and deposited in the developing oocyte. Since the yolk of a chicken egg contains over 90% of the biotin in an egg and all of the biotin is bound to biotin-binding protein, the function of biotin-binding protein is undoubtedly to transport biotin to the egg for future use by the developing embryo. Avidin is produced by the oviduct and in the egg it is presumed to deter microbial growth around the oocyte by sequestering biotin. Among the eggs examined, those from turkeys have the lowest amount of biotin-binding protein and the highest amount of avidin. Furthermore, the majority of the biotin in turkey eggs can be bound to avidin in the egg white, suggesting a nutritional role for avidin in turkeys. An assay has been developed to conveniently measure apo- and holobiotin-binding proteins.

Human α-chain globin messenger: Prediction of a nucleotide sequence

Abstract All nucleotide sequences consistent with the amino acid sequence of the α-chain of human hemoglobin were tested for their complementarity with a known 26-nucleotide sequence from the α-chain messenger. The region with the highest pairing potential is immediately adjacent to the known nucleotide sequence. The existence of this potential hairpin loop requires the specification of nucleotides in at least 6 degenerate positions.