DOCSLIB.ORG

HIV-1 Protease" Explored by Chemical Synthesis MANUEL BACA and STEPHEN B

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
HIV-1 Protease Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11638-11642, December 1993 Biochemistry Catalytic contribution of flap-substrate hydrogen bonds in "HIV-1 protease" explored by chemical synthesis MANUEL BACA AND STEPHEN B. H. KENT* The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037 Communicated by Harry B. Gray, August 9, 1993 ABSTRACT An analogue of "HIV-1 protease" was de- the inhibitor. In crystal structures of both types of aspartic signed in which the ability to donate important water-mediated proteinases, hydrogen bonds are observed between the pro- hydrogen bonds to substrate was precisely and directly deleted. tein backbone near the tip of the flap(s) and the carbonyl Chemical ligation ofunprotected peptide segments was used to oxygens flanking the pseudoscissile bond of bound, sub- synthesize this "backbone-engineered" enzyme. The function- strate-based peptide inhibitor. These flap-substrate hydro- ally relevant amide -CONH- linkage between residues Gly49- gen bonds have been implicated in an out-of-plane distortion le50 in each flap of the enzyme was replaced by an isosteric of the scissile amide bond, thereby facilitating attack by a thioester -COS- bond. The backbone-engineered enzyme had nucleophilic water molecule (6-9). However, while the amide normal substrate specificity and affinity (Kn). However, the -NH- of peptide bonds near the tip of the single flap in pepsin-like proteinases donate hydrogen bonds directly to the catalytic activity (kct) was reduced -3000-fold compared to substrate (6, 10), the corresponding hydrogen bonds in the the native amide bond-containing enzyme. Inhibition by the HIV-1 protease are contributed one from each flap and reduced peptide bond substrate analogue MVT-101 was unaf- appear to be mediated by a specific, tetrahedrally coordi- fected compared with native enzyme. By contrast, the normally nated internal water molecule, water 301 (5), poised between tight-binding hydroxyethylamine inhibitor JG-365 bound to the flaps and the inhibitor (Fig. 1A). This water molecule the backbone-engineered enzyme with an -2500-fold reduc- (which is not the nucleophilic water) has been observed in all tion in affinity. The reduced catalytic activity of the -Gly49- HIV-1 protease enzyme-inhibitor cocrystal structures so far *(COS)-lles- backbone-engineered enzyme analogue provides reported (11). This raises the question, are the water- direct experimental evidence to support the suggestion that mediated hydrogen bonds from the flaps to substrate/ backbone hydrogen bonds from the enzyme flaps to the sub- inhibitor important to the enzyme activity of the HIV-1 strate are important for the catalytic function of the HIV-1 protease? protease. We sought to experimentally address the role of the hydrogen bonds between enzyme flaps and the substrate in The mature proteins packaged within virions of the human the mode of action of the HIV-1 protease. An enzyme immunodeficiency virus (HIV) are initially translated as the analogue was designed in which the protein backbone would large precursor polyproteins gag and gag-pol. The role ofthe lack the ability to donate the flap hydrogen bonds observed aspartic proteinase from HIV-1 ("HIV-1 protease") is to in the x-ray crystal structure (5, 7, 8). A sulfur atom specif- proteolytically process these translation products to give ically substitutes for an -NH- in a single peptide bond in the functional core proteins and the enzymes required for viral backbone of each flap (Fig. 1B). This could be done with replication (1). surgical precision using the chemical dovetailing approach In contrast to the single-chain nonviral aspartic proteinases (13), which creates a backbone analogue structure at the such as pepsin, the HIV-1 protease has been characterized as ligation site. This report describes the synthesis and enzy- a homodimeric molecule (2). The monomer of the HIV-1 matic properties ofa "backbone-engineered" analogue ofthe protease consists of 99 amino acid residues, and each mono- HIV-1 protease, in which the functionally relevant amide mer contributes one ofthe two conserved active-site aspartyl bond between residues Gly49 and 11e50 has been replaced by residues. X-ray crystallography has revealed the HIV-1 pro- a thioester linkage. tease to be structurally related to the larger two-domain pepsin-like aspartic proteinases (2-4). Despite the difference MATERIALS AND METHODS in quaternary structure, the similarity between the two types of aspartic proteinases is immediately obvious upon super- Bromomethylvaleric Acid. (2R,3S)-2-Bromo-3-methylva- position of the molecular structures (2). leric acid (BrMV) was synthesized from D-allo-isoleucine An important structural feature of the aspartic proteinases [i.e., (2R,3S)-2-amino-3-methylvaleric acid] by in situ forma- is the 3-hairpin region known as the "flap". The flap is a tion of the diazonium salt in the presence ofbromide ion (14, mobile surface feature, characterized by high thermal factors 15). The crude reaction product was purified by reverse- in the crystal structures of the free enzyme (4). The ho- phase HPLC, and the identity of this product was confirmed modimeric HIV-1 protease molecule possesses two flaps, in by 'H NMR. The stereochemistry and chiral purity of this contrast to the single flap of the pepsin-like proteinases (2). compound was verified by synthesis of its diastereomer Upon binding inhibitor (and presumably substrate), the (2S,3S)-2-bromo-3-methylvaleric acid from L-isoleucine and flap(s) undergoes movement to interact with and lock the comparison oftheir 'H NMR spectra. Within the limits of 'H substrate into the active site, simultaneously desolvating the NMR sensitivity, BrMV was chirally pure. enzyme-bound ligand (4, 5). Peptide Synthesis. HIV-1 protease (SF2 strain) segments Potentially the most significant and intriguing difference 1-49 and 51-99 were synthesized as described elsewhere (13, between the HIV-1 protease and nonviral aspartic protein- ases is in the mode of interaction of the tip of the flap(s) with Abbreviations: HIV, human immunodeficiency virus; BrMV, (2R,3S)-2-bromo-3-methylvaleric acid; Boc, t-butoxycarbonyl; Aba, L-a-amino-n-butyric acid. The publication costs ofthis article were defrayed in part by page charge *To whom reprint requests should be addressed at: Mail Drop payment. This article must therefore be hereby marked "advertisement" CVN-6, The Scripps Research Institute, 10666 North Torrey Pines in accordance with 18 U.S.C. §1734 solely to indicate this fact. Road, La Jolla, CA 92037. 11638 Downloaded by guest on September 24, 2021 Biochemistry: Baca and Kent Proc. Natl. Acad. Sci. USA 90 (1993) 11639 A B FIG. 1. Hydrogen-bonding interactions be- tween the flap regions of the HIV-1 protease and a substrate-based inhibitor. (A) Hydrogen bonds from the enzyme backbone Ile50 -NH- in each flap to the inhibitor P2 and P1l carbonyls [notation of 11 \le50 li e5O' Schecter and Berger (12)] on either side of the isostere of the substrate scissile bond. The hydro- IS gen bonds are mediated by a specific, tetrahedrally coordinated water (water 301) observed in all HIV-1 protease-inhibitor complexes. The data were taken from the x-ray crystal structure of synthetic HIV-1 protease bound to the reduced isostere hexapeptide inhibitor MVT-101 (5). (B) Design of a backbone- engineered HIV-1 protease. This shows a hypothet- ical structure in which the single pertinent -CONH- in each flap has been converted to a -COS-, thus removing the ability to donate a hydrogen bond. Water 301 is modeled as hydrogen bonded only to the substrate-based inhibitor. [Note that in HIV-1 Asp25' Asp25 Asp25' Asp25 protease the scissile peptide bond is attacked by a water molecule (not water 301) located between the Asp25 and Asp25' side chains.] 16). The 51-99 segment contained L-a-amino-n-butyric acid extinction coefficient of 25,000 M-lcm-1 (20). Typically, (Aba) as an isosteric replacement for cysteine at positions 67 0.35 mg of folded enzyme analogue was obtained, reflecting and 95 (2). BrMV was coupled as the preformed symmetric a yield of 4.5% based on the starting peptide segments. anhydride (17) to the (51-99)HIV-1 protease segment, after Folded enzyme was stored in dialysis buffer at 4°C, and under first removing the His(dinitrophenyl) and Na-t-butoxycar- these conditions, no significant autolytic or chemical degra- bonyl (Boc) protecting groups from the resin-bound peptide dation ofthe enzyme was observed after a period of4 weeks. (16). Removal of remaining side chain protecting groups and The molecular mass of folded [(COS)49-50]HIV-1 protease from the resin was effected by treatment with dimer (hereafter referred to as backbone-engineered HIV-1 cleavage PR) was investigated by gel filtration using a Pharmacia hydrogen fluoride (16) to yield BrMV (51-99)HIV-1 protease. Superdex 75 column, running at 0.5 ml/min with 100 mM The peptide segment 1-49 was synthesized on a Boc-Gly-S- NaOAc/0.5 M NaCl, pH 5.3, as the mobile phase. The CH(phenyl)phenoxyacetamidomethyl resin (18). Cleavage concentration of enzyme as loaded onto the column was 0.4 and deprotection to yield the desired (1-49)-aCOSH segment ,uM, and protein peaks were detected by fluorescence at 335 was similarly performed after prior removal of the Na-Boc nm (excitation 280 nm). The observed molecular mass of group. backbone-engineered HIV-1 PR was determined from a plot Each peptide segment was purified by reverse-phase of log(molar mass) versus retention time, calibrated by coin- HPLC using linear gradients of acetonitrile in 0.1% aqueous jection of reference proteins. trifluoroacetic acid. Fractions collected were combined Enzymatic Properties. Comparison of the substrate speci- based on analysis by isocratic HPLC and ion-spray MS.
Load more

Recommended publications
  • John H. Northrop
    J OHN H . N ORTHROP T h e preparation of pure enzymes and virus proteins* Nobel Lecture, December 12, 1946 The problem of the chemical nature of the substances which control the reactions occurring in living cells has been a subject of research, and also of controversy, for nearly two hundred years. Before the eighteenth century these reactions were considered as "vital processes", outside the realm of experimental science. The work of Spallanzani, Payen and Persoz, Schwann, Kühne, and finally Buchner proved that many of these reactions could take place without living cells and were probably caused by the presence of small amounts of unstable and active substances, which Kühne called "enzymes". Berzelius, a century ago, pointed out that these enzymes were similar to the catalysts of the chemist and suggested that they be considered as special catalysts formed by the cells. This hypothesis was far ahead of its time and met with great opposition, since many workers considered that enzyme reac- tions differed qualitatively from ordinary chemical reactions. The work of Tamman, Arrhenius, Henri, Michaelis, Nelson, von Euler, Willstätter, War- burg, and other chemists, however, has shown that Berzelius’ viewpoint was correct and enzyme reactions are now considered a special kind of catalysis which does not differ qualitatively from other catalytic reactions. While the study of enzyme reactions made rapid progress all attempts to isolate an enzyme and so determine its chemical nature were unsuccessful until recently. The early workers were of the opinion that enzymes were probably pro- teins and in 1896 Pekelharing isolated a protein from gastric juice which he considered to be the enzyme pepsin.
    [Show full text]
  • DUAL ROLE of CATHEPSIN D: LIGAND and PROTEASE Martin Fuseka, Václav Větvičkab
    Biomed. Papers 149(1), 43–50 (2005) 43 © M. Fusek, V. Větvička DUAL ROLE OF CATHEPSIN D: LIGAND AND PROTEASE Martin Fuseka, Václav Větvičkab* a Institute of Organic Chemistry and Biochemistry, CAS, Prague, Czech Republic, and b University of Louisville, Department of Pathology, Louisville, KY40292, USA, e-mail: [email protected] Received: April 15, 2005; Accepted (with revisions): June 20, 2005 Key words: Cathepsin D/Procathepsin D/Cancer/Activation peptide/Mitogenic activity/Proliferation Cathepsin D is peptidase belonging to the family of aspartic peptidases. Its mostly described function is intracel- lular catabolism in lysosomal compartments, other physiological effect include hormone and antigen processing. For almost two decades, there have been an increasing number of data describing additional roles imparted by cathepsin D and its pro-enzyme, resulting in cathepsin D being a specific biomarker of some diseases. These roles in pathological conditions, namely elevated levels in certain tumor tissues, seem to be connected to another, yet not fully understood functionality. However, despite numerous studies, the mechanisms of cathepsin D and its precursor’s actions are still not completely understood. From results discussed in this article it might be concluded that cathepsin D in its zymogen status has additional function, which is rather dependent on a “ligand-like” function then on proteolytic activity. CATHEPSIN D – MEMBER PRIMARY, SECONDARY AND TERTIARY OF ASPARTIC PEPTIDASES FAMILY STRUCTURES OF ASPARTIC PEPTIDASES Major function of cathepsin D is the digestion of There is a high degree of sequence similarity among proteins and peptides within the acidic compartment eukaryotic members of the family of aspartic peptidases, of lysosome1.
    [Show full text]
  • Hydrolysis of -Lactalbumin by Chymosin and Pepsin. Effect Of
    Hydrolysis of α-lactalbumin by chymosin and pepsin. Effect of conformation and pH G. Miranda, G. Hazé, Non Renseigné To cite this version: G. Miranda, G. Hazé, Non Renseigné. Hydrolysis of α-lactalbumin by chymosin and pepsin. Effect of conformation and pH. Le Lait, INRA Editions, 1989, 69 (6), pp.451-459. hal-00929176 HAL Id: hal-00929176 https://hal.archives-ouvertes.fr/hal-00929176 Submitted on 1 Jan 1989 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Lait (1989) 69. 451-459 451 © Elsevier/INRA Original article Hydrolysis of œ-lactalburnin by chymosin and pepsin. Effect of conformation and pH G. Miranda, G. Hazé, P.Scanff and J.P.Pélissier INRA, station de recherches laitières, 78350 Jouy-en-Josas, France (received 21 March 1989. accepted 26 June 1989) Summary - The correlation between change of conformation of a-Iactalbumin and its degradation by gastric enzymes was verified. With citrate buffer (0.1 M), the modification of a-Iactalbumin confor- mation occurred when the pH value was below pH 4.0. This conformational change was influenced by buffer composition and ionic strength. However, the presence of EDTA in the butter did not modi- fy the pH value at which the change of conformation of the protein occurred.
    [Show full text]
  • Structure of the Human Renin Gene
    Proc. Nati. Acad. Sci. USA Vol. 81, pp. 5999-6003, October 1984 Biochemistry Structure of the human renin gene (hypertension/aspartyl proteinase/nucleotide sequence/splice junction) HITOSHI MIYAZAKI*, AKIYOSHI FUKAMIZU*, SHIGEHISA HIROSE*, TAKASHI HAYASHI*, HITOSHI HORI*, HIROAKI OHKUBOt, SHIGETADA NAKANISHIt, AND KAZUO MURAKAMI** *Institute of Applied Biochemistry, University of Tsukuba, Ibaraki 305, Japan; and tInstitute for Immunology, Kyoto University Faculty of Medicine, Kyoto 606, Japan Communicated by Leroy Hood, June 27, 1984 ABSTRACT The human renin gene was isolated from a between the intron-exon organization of the gene and the Charon 4A human genomic library and characterized. The tertiary structure of the protein. gene spans about 11.7 kilobases and consists of 10 exons and 9 introns that map at points that could be variable surface loops MATERIALS AND METHODS of the enzyme. The complete coding regions, the 5'- and 3'- Materials. All restriction enzymes were obtained from flanking regions, and the exon-intron boundaries were se- either New England Biolabs or Takara Shuzo (Kyoto, Ja- quenced. The active site aspartyl residues Asp-38 and Asp-226 pan). Escherichia coli alkaline phosphatase and T4 DNA li- are encoded by the third and eighth exons, respectively. The gase were from Takara Shuzo. [_y-32P]ATP (>5000 Ci/mmol; extra three amino acids (Asp-165, Ser-166, Glu-167) that are 1 Ci = 37 GBq) and [a-32P]dCTP (=3000 Ci/mmol) were not present in mouse renin are encoded by the separate sixth from Amersham. exon, an exon as small as 9 nucleotides. The positions of the Screening. A human genomic library, prepared from partial introns are in remarkable agreement with those in the human Alu I and Hae III digestion and ligated into the EcoRI arms pepsin gene, supporting the view that the genes coding for of the X vector Charon 4A, was kindly provided by T.
    [Show full text]
  • Proteolytic Cleavage—Mechanisms, Function
    Review Cite This: Chem. Rev. 2018, 118, 1137−1168 pubs.acs.org/CR Proteolytic CleavageMechanisms, Function, and “Omic” Approaches for a Near-Ubiquitous Posttranslational Modification Theo Klein,†,⊥ Ulrich Eckhard,†,§ Antoine Dufour,†,¶ Nestor Solis,† and Christopher M. Overall*,†,‡ † ‡ Life Sciences Institute, Department of Oral Biological and Medical Sciences, and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada ABSTRACT: Proteases enzymatically hydrolyze peptide bonds in substrate proteins, resulting in a widespread, irreversible posttranslational modification of the protein’s structure and biological function. Often regarded as a mere degradative mechanism in destruction of proteins or turnover in maintaining physiological homeostasis, recent research in the field of degradomics has led to the recognition of two main yet unexpected concepts. First, that targeted, limited proteolytic cleavage events by a wide repertoire of proteases are pivotal regulators of most, if not all, physiological and pathological processes. Second, an unexpected in vivo abundance of stable cleaved proteins revealed pervasive, functionally relevant protein processing in normal and diseased tissuefrom 40 to 70% of proteins also occur in vivo as distinct stable proteoforms with undocumented N- or C- termini, meaning these proteoforms are stable functional cleavage products, most with unknown functional implications. In this Review, we discuss the structural biology aspects and mechanisms
    [Show full text]
  • Pepsin Certificate of Analysis 9PIV1959
    Certificate of Analysis Pepsin: Part No. Size Part# 9PIV1959 V195A 250mg Revised 8/16 Description: Pepsin preferentially cleaves at the C-terminus of phenylalanine, leucine, tyrosine and tryptophan (1–4). This protease can be used alone or in combination with other proteases for protein analysis by mass spectrometry and other applications. Biological Source: Porcine stomach . Molecular Weight: 34.6kDa . Form: Lyophilized . Storage Conditions: See the Product Information Label for storage conditions and expiration date. *AF9PIV19590816V1959* Optimal pH: 1.0–3.0 (4–6). AF9PIV19590816V1959 Activators: Hydrochloric acid (HCl), trifluoroacetic acid (TFA). Inactivators: pH greater than 6.0. Usage Notes: 1. Resuspend Pepsin in double-distilled water (pH 5.5 or lower) to a final concentration of 1mg/ml. Store reconstituted Pepsin at 4°C for up to 1 month. 2. Specificity for cleavage at Phe and Leu is best at pH 1.0 and decreased above pH 2.0. Pepsin irreversibly inactivates above pH 6.0. Promega Corporation 2800 Woods Hollow Road Madison, WI 53711-5399 USA Telephone 608-274-4330 Toll Free 800-356-9526 Fax 608-277-2516 Quality Control Assays Internet www.promega.com This lot passes the following Quality Control specifications: Activity: Digestion reactions using insulin as a substrate are performed at a protease:substrate ratio of 1:20 and analyzed by PRODUCT USE LIMITATIONS, WARRANTY, DISCLAIMER reverse-phase HPLC. Intact substrate is undetectable after incubation for 15 minutes at 37°C. Promega manufactures products for a number of intended uses. Please refer to the product label for the intended use statements for specific products. Usage Information on Back Promega products contain chemicals which may be harmful if misused.
    [Show full text]
  • Cleavage of Honeybee Prepromelittin by an Endoprotease from Rat Liver Microsomes: Identification of Intact Signal Peptide
    Proc. Natl. Acad. Sci. USA Vol. 79, pp. 2260-2263, April 1982 Biochemistry Cleavage of honeybee prepromelittin by an endoprotease from rat liver microsomes: Identification of intact signal peptide (precursor processing/signal peptidase/membrane protein/melittin) CHRISTA MOLLAY, ULRIKE VILAS, AND GUNTHER KREIL Institute for Molecular Biology, Austrian Academy of Sciences, Billrothstrasse 11, A5020 Salzburg, Austria Communicated by Paul D. Boyer, January 19, 1982 ABSTRACT It has previously been shown that rat liver mi- tivities, on a sucrose-deoxycholate gradient. This endoprotease crosomes contain a proteolytic enzyme that cleaves honeybee pre- cleaves prepromelittin at a single peptide bond, the pre- promelittin to yield promelittin. This enzyme has now been fur- pro junction. The-present communication describes the isola- ther purified by centrifugation on a sucrose-deoxycholate gradient tion ofthe second reaction product, intact signal peptide, from and then reconstituted into phospholipid vesicles. Incubation of such in vitro assays. prepromelittin with vesicles in the presence of melittin yields, in addition to promelittin, a hydrophobic peptide. The latter could be isolated by extraction with 1-butanol and paper electrophoresis MATERIALS AND METHODS in 30% formic acid and was shown to be intact signal peptide by analysis of peptic fragments and automated Edman degradation. Tritium-labeled amino acids ofthe highest specific activity cur- The microsomal enzyme is thus an endoprotease that hydrolyzes rently available were purchased from the Radiochemical Centre prepromelittin exclusively at the pre-pro junction. The precision (Amersham, England). Phospholipids were obtained from Lipid of this cleavage of an insect preprotein by a rat liver enzyme in- Products (Nutfield Nurseries, England), synthetic dipeptides dicates that we are dealing with the ubiquitous eukaryotic signal were from Bachem AG (Bubendorf, Switzerland), and other peptidase.
    [Show full text]
  • Protease Inhibitors in Human Milk
    Pediat. Res 13: 969-972 (1979) Chymotrypsin protease inhibi- elastase tors human milk trypsin Protease Inhibitors in Human Milk TOR LINDBERG Departments of Pediatrics and Experimental Research, Malmo General Hospital, University of Lund, Malmo, Sweden Summary standard serum for the measurements of the concentration of a,- antitrypsin and antichymotrypsin were gifts of Dr P. Fernlund, Protease inhibitors (inhibiting trypsin, chymotrypsin, and elas- Department of Clinical Chemistry, Malmo General Hospital. A tase) were demonstrated in human milk from birth to 4 months standard pool of serum from 1000 donors was regarded as 100'70 after delivery. No pepsin inhibitor was found. The protease inhib- = about 1.35 g a,-antitrypsin/liter and 0.5 g antichymotrypsin/ itors were localized in the al-region-inhibiting trypsin, chymo- liter. trypsin, and elastase-and in a more cathodal region-inhibiting Chemicals: Agarose (Miles-Seravac, Maidenhead, England) ca- chymotrypsin-in agarose gel electrophoresis of human milk. al- sein (BDH), elastin (Worthington), N-benzoyl-DL-arginine-p-ni- antitrypsin and antichymotrypsin were demonstrated by crossed troanilide (BAPNA) (Sigma), bovine trypsin (EC 3.4.4.4.) (Fluka), immunoelectrophoresis. Electroimmunoassay showed the concen- a-chymotrypsin (EC 3.4.4.5) (Fluka), pepsin (EC 3.4.23.1) (Fluka), tration of a,-antitrypsin in 1st day milk to be 109% and the and elastase (EC 3.4.4.7) (Sigma). concentration of antichymotrypsin was 116% of that of adult serum. The concentrations d&;eased during the 1st wk; from 1 wk to 4 months they were 1.6% for a,-antitrypsin and 3.84% for METHODS antichymotrypsin of those of adult serum.
    [Show full text]
  • Q1.Some Students Investigated the Effect of Ph on the Digestion of Boiled Egg White by an Enzyme Called Pepsin
    Kingsmead Technology College Q1.Some students investigated the effect of pH on the digestion of boiled egg white by an enzyme called pepsin. Egg white contains protein. The students: • put a glass tube containing boiled egg white into a test tube • added a solution containing pepsin at pH 7 • set up six more tubes with solutions of pepsin at different pH values • left the test tubes for 24 hours at room temperature. The image below shows one of the test tubes, at the start and at the end of the 24 hours. At start 24 hours later (a) (i) Name the product of protein digestion. ............................................................................................................... (1) (ii) What type of enzyme digests protein? Tick ( ) one box. amylase lipase protease Page 1 Kingsmead Technology College (1) (b) The egg white in each tube was 50 mm long at the start of the investigation. The table below shows the students’ results. Length in mm of boiled pH egg white after 24 hours 1 38 2 20 3 34 4 45 5 50 6 50 7 50 (i) At which pH did the pepsin work best? pH .................................. (1) (ii) The answer you gave in part (b)(i) may not be the exact pH at which pepsin works best. What could the students do to find a more accurate value for this pH? ............................................................................................................... ............................................................................................................... ..............................................................................................................
    [Show full text]
  • Ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric)
    Version 2a Last updated 15 February 2021 ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric) For the measurement of pepsin in biological samples. This product is for research use only and is not intended for diagnostic use. ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric) Table of Contents 1. Overview 3 2. Materials Supplied and Storage 4 3. Materials Required, Not Supplied 5 4. General guidelines, precautions, and troubleshooting 6 5. Reagent Preparation 7 6. Standard Preparation 8 7. Sample Preparation 9 8. Assay Procedure 10 9. Data Analysis 12 10. Typical Data 13 11. Notes 15 ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric) 1. Overview Pepsin/Pepsinogen Assay Kit (Fluorometric) (ab238722) is a homogenous assay that allows for quantification of pepsin activity in gastric tissues (stomach, duodenum etc.) and various biological fluids (serum/plasma, gastric juice, vomit). The assay utilizes a synthetic peptide substrate bearing both a fluorophore and a fluorescence quencher. Upon cleavage by pepsin, the fluorophore- bearing peptide fragment is unquenched to produce a bright fluorescent signal (Ex/Em = 328/418 nm). Lysosomal aspartic proteases in the peptidase A1 family (Cathepsin D and E) do not interfere with the assay. The assay is rapid, simple to perform and is vastly more sensitive than the classical hemoglobin degradation assay, with a detection limit of 500 μU pepsin activity per well. Pepsin / acidic conditions Peptide substrate Fluorescent peptide fragment (Quenched) (Ex/Em = 328/418 nm) Prepare tissue / serum / plasma sample and Pepsin Positive Control. Prepare Standard Curve. Prepare Reaction Mix, Inhibitor Reaction Mix, Background Control Mix and Positive Control Reaction Mix. Start reaction by adding 20 μL diluted Substrate working solution to each well (apart from Standard Curve).
    [Show full text]
  • Gastric Inhibitory Polypeptide (GIP)*
    J Clin Pathol: first published as 10.1136/jcp.s1-8.1.31 on 1 January 1978. Downloaded from J. clin. Path., 33, Suppl. (Ass. Clin. Path.), 8, 31-37 Gastric inhibitory polypeptide (GIP) * D. L. SARSON From the Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 OHS As long ago as 1930, Kosaka and Lim proposed a sample with 8. 0 M urea. Thus, this peak may humoral agent capable of inhibiting gastric acid represent a protein/peptide complex. secretion after a meal. They coined the term entero- (2) A large molecular form which may correspond gastrone. Using crude preparations of cholecysto- to either a 'big GIP' or a precursor pro-GIP. kinin-pancreozymin (CCK PZ) in dogs they were (3) A 5000 dalton molecular form. This peak elutes able to inhibit the acid secretion normally stimulated in the same position as porcine standard and by a meat meal or histamine. They went on to 1251-labelled GIP. experiment with duodenal extracts, prepared after The exact nature and properties of these different the instillation into the duodenum of olive oil, molecular species require further analysis to deter- and found a similar effect to that seen with crude mine which form or forms of GIP are biologically CCK PZ. The effects of similar crude preparations active. ofCCK PZ in the dog were confirmed Brown On the basis of several by and amino-acid sequence copyright. Pederson in 1970, but further purification of this homologies GIP has been placed in the classical material led to a diminution of the acid inhibitory secretin-glucagon-VIP family of peptides.
    [Show full text]
  • Engineering the Residual Side Chains of HAP Phytases to Improve Their
    www.nature.com/scientificreports OPEN Engineering the residual side chains of HAP phytases to improve their pepsin resistance and catalytic Received: 04 October 2016 Accepted: 06 January 2017 efficiency Published: 10 February 2017 Canfang Niu, Peilong Yang, Huiying Luo, Huoqing Huang, Yaru Wang & Bin Yao Strong resistance to proteolytic attack is important for feed enzymes. Here, we selected three predicted pepsin cleavage sites, L99, L162, and E230 (numbering from the initiator M of premature proteins), in pepsin-sensitive HAP phytases YkAPPA from Yersinia kristensenii and YeAPPA from Y. enterocolitica, which corresponded to L99, V162, and D230 in pepsin-resistant YrAPPA from Y. rohdei. We constructed mutants with different side chain structures at these sites using site-directed mutagenesis and produced all enzymes in Escherichia coli for catalytic and biochemical characterization. The substitutions E230G/A/P/R/S/T/D, L162G/A/V, L99A, L99A/L162G, and L99A/L162G/E230G improved the pepsin resistance. Moreover, E230G/A and L162G/V conferred enhanced pepsin resistance on YkAPPA and YeAPPA, increased their catalytic efficiency 1.3–2.4-fold, improved their stability at 60 °C and pH 1.0–2.0 and alleviated inhibition by metal ions. In addition, E230G increased the ability of YkAPPA and YeAPPA to hydrolyze phytate from corn meal at a high pepsin concentration and low pH, which indicated that optimization of the pepsin cleavage site side chains may enhance the pepsin resistance, improve the stability at acidic pH, and increase the catalytic activity. This study proposes an efficient approach to improve enzyme performance in monogastric animals fed feed with a high phytate content.
    [Show full text]