Publisher Summary This chapter explores that the changes that take place in the protein molecules during denaturation constitute one of the most interesting and complex classes of reactions that can be found either in nature or in the laboratory These reactions are important because of the information they can provide about the more intimate details of protein structure and function They are also significant because they challenge the chemist with a difficult area for the application of chemical principles The chapter reviews that the denaturation is a process in which the spatial arrangement of the polypeptide chains within the molecule is changed from that typical of the native protein to a more disordered arrangement The chapter also discusses the classification of protein structures: primary, secondary, and tertiary structures The primary structure is that expressed by the structural chemical formula and depends entirely on the chemical valence bonds that the classical organic chemist would write down for the protein molecule The secondary structure is the configuration of the polypeptide chain that results from the satisfaction of the hydrogen bonding potential between the peptide N-H and C=O groups The tertiary structure is the pattern according to which the secondary structures are packed together within the native protein molecule The term “denaturation” as used in this chapter is indented to include changes in both the secondary and tertiary structures

Some factors in the interpretation of protein denaturation.

9-Fluorenylmethoxycarbonyl (Fmoc) amino acids were first used for solid phase peptide synthesis a little more than a decade ago. Since that time, Fmoc solid phase peptide synthesis methodology has been greatly enhanced by the introduction of a variety of solid supports, linkages, and side chain protecting groups, as well as by increased understanding of solvation conditions. These advances have led to many impressive syntheses, such as those of biologically active and isotopically labeled peptides and small proteins. The great variety of conditions under which Fmoc solid phase peptide synthesis may be carried out represents a truly "orthogonal" scheme, and thus offers many unique opportunities for bioorganic chemistry.

Solid phase peptide synthesis utilizing 9‐fluorenylmethoxycarbonyl amino acids

Though the structures presented in crystallographic models of macromolecules appear to possess rock-like solidity, real proteins and nucleic acids are not particularly rigid. Most structural work to date has centred upon the native state of macromolecules, the most probable macromolecular form. But the native state of a molecule is merely its most abundant form, certainly not its only form. Thermodynamics requires that all other possible structural forms, however improbable, must also exist, albeit with representation corresponding to the factor exp( — Gi/RT) for each state of free energy Gi (see Moelwyn-Hughes, 1961), and one appreciates that each molecule within a population of molecules will in time explore the vast ensemble of possible structural states.

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/S0033583500005217

Hydrogen exchange and structural dynamics of proteins and nucleic acids

Polyphenol oxidases in plants

When producing hydrolyzed proteins, it is important to determine the degree of hydrolysis (DH). The trinitro-benzene-sulfonic acid (TNBS) method is well established with regard to enzymatic hydrolysis. However, this method is laborious, cannot be used to follow a hydrolysis reaction continuously, and includes hazardous and unstable chemicals. This paper describes a method based on the reaction of primary amino groups with o- phthaldialdehyde (OPA). The conclusion is that the OPA method of analyzing the DH of protein hydrolyses is more accurate, is easier and faster to carry out, has a broader application range, and is environmentally safer than the TNBS method.

/pdf/improved-method-for-determining-food-protein-degree-of-4obrs5c68s.pdf

Improved Method for Determining Food Protein Degree of Hydrolysis

[11] The subtilisins

Aspergillus niger glucoamylases GI and GII (EC 3213) were isolated from a commercial enzyme preparation by ammonium sulfate precipitation followed by DEAE-cellulose ion exchange chromatography Both enzymes consist of a single glycosylated polypeptide chain The molecular weights of GI and GII were determined by sedimentation equilibrium ultracentrifugation to 52,000 and 46,000, respectively, and by molecular sieving to 65,000 and 55,000 The amino acid compositions of GI and GII were very similar Furthermore, the N-terminal amino acid sequence of the intact GI and GII as well as of their cyanogen fragments were identical, suggesting great homology in the primary structure of the two forms In addition the digests of GI and GII produced respectively by Armillaria mellea protease, Staphylococcus aureus V8 protease, and submaxillary protease were analyzed by high pressure gel permeation chromatography The elution profiles were also consistent with GI and GII having similar polypeptide chains However, digestion with carboxypeptidase Y showed different C-terminal residues of the two forms

https://link.springer.com/content/pdf/10.1007/BF02907797.pdf

Characterization of two forms of glucoamylase from aspergillus niger

The pH-stat and its use in biochemistry.

Carboxypeptidase Y from bakers’ yeast has been purified in high yields by affinity chromatography. The affinity gel was prepared by coupling the specific inhibitor p-aminobenzylsuccinic acid via an azo linkage to Sepharoseglycyl-tyrosine. This affinity gel was able to bind carboxypeptidase Y specifically and quantitatively from a crude yeast autolysate. The isolated enzyme appeared homogeneous by gel electrophoresis and ultracentrifugation, while isoelectric focusing revealed the presence of two components with isoelectric points of pH 3.56 and 3.66, respectively. Small differences in amino acid composition and enzymatic properties between the enzyme from danish yeast and the corresponding enzyme isolated from Fleichmann yeast suggested the existence of more than one form of this enzyme.

https://link.springer.com/content/pdf/10.1007/BF02908689.pdf

Martin Ottesen

Papers

[11] The subtilisins

Characterization of two forms of glucoamylase from aspergillus niger

The pH-stat and its use in biochemistry.

Isolation of carboxypeptidase Y by affinity chromatography

Studies on the Structural Basis of Ribonuclease Activity